Modified T lymphocytes comprising a BAFF antibody-inducible caspase and methods of apoptosis

ABSTRACT

Provided herein are cells, e.g., T cells expressing artificial cell death polypeptides that cause death of a cell, e.g., cells (e.g., T lymphocytes) expressing the cell death polypeptide, when the cell death polypeptide is multimerized or dimerized. Also provided herein is use of such cells, e.g., T lymphocytes, to treat diseases such as cancer.

This application is a divisional of U.S. patent application Ser. No.14/775,891, which is a U.S. National Stage of International PatentApplication No. PCT/US2014/027039, filed on Mar. 14, 2014, which claimspriority to the benefit of U.S. Provisional Patent Application No.61/794,294, filed Mar. 15, 2013, the disclosure of each of which isincorporated herein by reference herein in its entirety.

A Sequence Listing in Computer Readable Form (“CRF”), which is an ASCIItext file of 3,161 bytes in size entitled 14247-375-999_SEQ_LISTING.txt, created on Feb. 7, 2019, which is being filedconcurrently with the instant application via EFS-Web, is incorporatedby reference herein in its entirety.

1. FIELD

The disclosure herein relates to the field of immunology, and morespecifically, to the modification of T lymphocytes or other immunecells.

2. BACKGROUND

T lymphocytes recognize and interact with specific antigens, includingtumor-associated or tumor-specific antigens. Because T lymphocytes areable to kill tumor cells, the last 25 years has seen a great deal ofinterest in targeting tumor cells with T lymphocytes, eitherantigen-specific T lymphocytes, or T lymphocytes genetically modified toexpress one or more chimeric antigen receptors (CARs; see, e.g., Eshhar,U.S. Pat. No. 7,741,465; Eshhar, U.S. Patent Application Publication No.2012/0093842). However, given the ability of T lymphocytes to kill notonly tumor cells displaying a certain antigen but normal cellsdisplaying the same antigen, it is desirable to incorporate into the Tlymphocytes a safety mechanism that enables rapid killing of the cellsafter administration to a patient should off-target effects provedeleterious to the patient.

While a system to kill T cells has described (Straathof et al. (2005)Blood 105(11):4247-4254), this system was dependent upon specific anddifficult-to-make protein modifications, rendering the systemundesirable for practical use. As such, there exists a need in the artfor a safety system to rapidly kill therapeutic T lymphocytes that arerelatively simple and straightforward to construct. T lymphocytescomprising such a safety system are provided herein.

3. SUMMARY

Provided herein are genetically modified cells, for example immunecells, such as T lymphocytes, e.g., human T lymphocytes, that comprisean artificial multimerizable, e.g., dimerizable, polypeptide (referredto herein as a “cell death polypeptide”) that, when multimerized, e.g.,dimerized, by a multimerizing agent, e.g., dimerizing agent, generatesan apoptosis-inducing signal in a cell, e.g., a T lymphocyte, thatexpresses the polypeptide, resulting in cell death, e.g., via apoptosis.Without wishing to be bound by any particular mechanism or theory, it isthought that when a sufficient number of a plurality of cell deathpolypeptides of the cell are multimerized, e.g., dimerized, that theaggregate apoptosis-inducing signal thereby generated is sufficient tokill the cell, e.g., cause the cell to undergo apoptosis.

The cell death polypeptides provided herein may be used in conjunctionwith any cells, in particular, any mammalian cells, for example, anyhuman cells. For example, such cell death polypeptides provide, forexample, a useful safety feature for cell therapeutics. As such, thecell death polypeptides can, for example, be important for a drugproduct comprising a cell therapeutic, e.g., a chimeric antigenreceptor-expressing CAR T lymphocytes, because the cell deathpolypeptides enable rapid killing of the cell therapeutic, e.g., the Tlymphocytes, should such rapid killing become desirable, e.g., in theevent administration of the cells causes any unwanted or deleteriouseffects in a patient receiving them, or if the presence of the celltherapeutic, e.g., the T lymphocytes, in a subject is no longernecessary. Thus, in certain embodiments, the cell death polypeptidesprovided herein can be used in conjunction with any administrable cells,for example cell therapeutics, such as mammalian cell therapeutics,e.g., human cell therapeutics. Non-limiting examples of cells in whichthe cell death polypeptides and multimerizing or dimerizing agents maybe used include, but are not limited to, natural killer (NK) cells,dendritic cells (DC), placental stem cells (e.g., the placental stemcells disclosed in U.S. Pat. Nos. 7,468,276; 8,057,788 and 8,202,703,the disclosures of which are hereby incorporated by reference in theirentireties), mesenchymal-like stem cells from umbilical cord blood,placental blood, peripheral blood, bone marrow, dental pulp, adiposetissue, osteochondral tissue, and the like; embryonic stem cells,embryonic germ cells, neural crest stem cells, neural stem cells, anddifferentiated cells (e.g., fibroblasts, etc.). The cell deathpolypeptides, and multimerizing or dimerizing agents, may also be usedin tumor cell lines, e.g., for animal model experimental purposes.

Typically, the cell death polypeptide is multimerizable or dimerizableusing an administrable multimerizing or dimerizing agent, e.g., a smallmolecule, polypeptide (other than the cell death polypeptide) such as anantibody, an oligonucleotide, or a polysaccharide. The cell deathpolypeptides do not comprise a FK506 binding protein (FKBP), functionalportion thereof, of modified form thereof, and the multimerizing agentor dimerizing agent is not an FKBP ligand.

In a first aspect, provided herein is a cell, e.g., a T lymphocyte,comprising a cell death polypeptide comprising an apoptosis-inducingdomain, wherein said cell death polypeptide is multimerizable using amultimerizing agent, wherein when said multimerizing agent multimerizessaid polypeptide, an apoptosis-inducing signal is generated in saidcell. In a specific embodiment, said multimerizing agent is a dimerizingagent; that is, the multimerizing agent causes the cell deathpolypeptide to dimerize. In another specific embodiment, when saiddimerizing agent dimerizes said polypeptide, an apoptosis-inducingsignal is generated in said cell.

In certain embodiments, said cell death polypeptide is a transmembranepolypeptide comprising an extracellular domain, a transmembrane domain,and an intracellular domain comprising said apoptosis-inducing domain.In particular embodiments, the apoptosis-inducing domain of the celldeath polypeptide is or comprises a caspase, e.g., caspase 9, caspase 8,or caspase 3, for example a human caspase 9, caspase 8, or caspase 3.

In certain embodiments, the dimerizing agent is a polypeptide comprisingat least two sites that specifically bind to a cell death polypeptide,e.g., an extracellular domain of a cell death polypeptide. In particularembodiments, the polypeptide is an antibody, e.g., an antibodycomprising at least two epitope or mimotope binding sites. In certainembodiments, only the antigen binding domain of an antibody is used as amultimerizing or dimerizing agent. In certain embodiments, anextracellular domain of a cell death polypeptide comprises at least oneepitope or mimotope to which the antibody specifically binds. Inparticular embodiments, the antibody is a bispecific antibody comprisingtwo different epitope or mimotope binding sites that bind two differentepitopes or mimotopes present on an extracellular domain of a cell deathpolypeptide. In certain embodiments, the antibody is an IgG or an IgMantibody. In a particular embodiment, an antibody useful as amultimerizing or dimerizing agent is one that has been approved by theUnited States Food and Drug Administration for any use.

In one embodiment, an antibody useful as a multimerizing or dimerizingagent is one that specifically binds to a CD20 epitope or mimotope,e.g., a human CD20 epitope or mimotope, and an extracellular domain of acell death polypeptide comprises a CD20 epitope or mimotope to which theantibody specifically binds. In certain specific embodiments, theantibody is rituximab and an extracellular domain of a cell deathpolypeptide comprises a CD20 epitope or a CD20 mimotope thatspecifically binds to said rituximab. In another specific embodiment,the antibody is tositumumab and an extracellular domain of a cell deathpolypeptide comprises a CD20 epitope or a CD20 mimotope thatspecifically binds to said tositumumab. In yet another embodiment, theantibody is ibritumomab and an extracellular domain of a cell deathpolypeptide comprises a CD20 epitope or a CD20 mimotope thatspecifically binds to said ibritumomab. In still another embodiment, theantibody is ofatumumab and an extracellular domain of a cell deathpolypeptide comprises a CD20 epitope or a CD20 mimotope thatspecifically binds to said ofatumumab.

In another specific embodiment, the antibody is alemtuzumab and anextracellular domain of a cell death polypeptide comprises a CD52epitope or a CD52 mimotope that specifically binds to said alemtuzumab.In yet another embodiment, the antibody is basiliximab and anextracellular domain of the cell death polypeptide comprises a CD25epitope or a CD25 mimotope that specifically binds to said basiliximab.In another embodiment, the antibody is daclizumab and an extracellulardomain of a cell death polypeptide comprises a CD25 epitope or a CD25mimotope that specifically binds to said daclizumab. In still anotherembodiment, the antibody is brentuximab and an extracellular domain of acell death polypeptide comprises a CD30 epitope or a CD30 mimotope thatspecifically binds to said brentuximab. In another embodiment, theantibody is belimumab and an extracellular domain of a cell deathpolypeptide comprises a B-cell activating factor (BAFF) epitope or aBAFF mimotope that specifically binds to said belimumab. In anotherembodiment, the antibody is cetuximab and an extracellular domain of acell death polypeptide comprises an epidermal growth factor receptor(EGFR) epitope or an EGFR mimotope that specifically binds to saidcetuximab. In yet another embodiment, the antibody is panitumumab and anextracellular domain of a cell death polypeptide comprises an epidermalgrowth factor receptor (EGFR) epitope or an EGFR mimotope thatspecifically binds to said panitumumab. In another embodiment, theantibody is efalizumab and an extracellular domain of a cell deathpolypeptide comprises an epitope of CD11a or a mimotope of CD11a thatspecifically binds to said efalizumab. In still another embodiment, theantibody is ipilimumab and an extracellular domain of a cell deathpolypeptide comprises a CD152 epitope or CD152 mimotope thatspecifically binds to said ipilimumab. In still another embodiment, theantibody is natalizumab and an extracellular domain of a cell deathpolypeptide comprises an epitope of alpha-4 integrin or a mimotope ofalpha 4 integrin that specifically binds to said natalizumab. In anotherembodiment, the antibody is basiliximab and an extracellular domain of acell death polypeptide comprises a CD25 epitope or CD25 mimotope thatspecifically binds to said basiliximab.

In certain embodiments, when a multimerizing agent or a dimerizing agentbinds to at least two cell death polypeptides, dimerization ormultimerization of the cell death polypeptides occurs, e.g.,dimerization or multimerization of the cell death polypeptides occurs.In certain embodiments, an extracellular domain of a cell deathpolypeptide is or comprises a receptor or a ligand-binding portionthereof. In a specific embodiment, a multimerizing agent or dimerizingagent is or comprises at least two ligands for said receptor or ligandbinding portion thereof. In another specific embodiment, saidmultimerizing agent or dimerizing agent binds to said receptor or saidligand binding portion thereof on two of the cell death polypeptides,and said polypeptides are multimerized or dimerized, e.g., theintracellular domains of said polypeptides are multimerized ordimerized. In particular embodiments the cell death polypeptidescomprise intracellular domains comprising a caspase domain, andmultimerization or dimerization of the caspase domains occurs. Inspecific embodiments, said multimerization or dimerization, for example,multimerization or dimerization of intracellular domains, e.g.,multimerization or dimerization of caspase domains, initiates anapoptosis-inducing signal in said cell, e.g., T lymphocyte.

In specific embodiments, when an antibody specifically binds to anepitope or mimotope of at least two cell death polypeptides,dimerization of the cell death polypeptides occurs, e.g., dimerizationof the intracellular domains of the cell death polypeptides occurs. Inparticular embodiments the cell death polypeptides compriseintracellular domains comprising a caspase domain, and dimerization ofthe caspase domains occurs. In specific embodiments, said dimerization,for example, dimerization of intracellular domains, e.g., dimerizationof caspase domains, initiates an apoptosis-inducing signal in said cell,e.g., T lymphocyte.

In certain other embodiments of the cell, e.g., T lymphocyte, saidextracellular domain of the cell death polypeptide comprises a ligandfor a receptor. In a specific embodiment, said multimerizing agent ordimerizing agent comprises at least two receptors or ligand-bindingportions thereof that bind to said ligand. In a specific embodiment,when said multimerizing agent or dimerizing agent binds to said receptoror said ligand binding portion thereof on at least two of the cell deathpolypeptides, said polypeptides are multimerized or dimerized. In aspecific embodiment, when the cell death polypeptides are multimerizedor dimerized, an apoptosis-inducing signal is generated in said cell.

In certain other embodiments, an extracellular domain of a cell deathpolypeptide comprises an artificial oligonucleotide sequence. Forexample, in particular embodiments, a modified cell, e.g., T lymphocyte,comprises a cell death polypeptide comprising an extracellular domainthat comprises an artificial oligonucleotide sequence. In a specificembodiment, a multimerizing or dimerizing agent is or comprises at leastone multimerizing or dimerizing oligonucleotide comprising a firstoligonucleotide and a second oligonucleotide, optionally joined by alinker, wherein said first oligonucleotide and said secondoligonucleotide are complementary to said artificial oligonucleotidesequence. In certain specific embodiments, said first oligonucleotideand said second oligonucleotide have the same sequence. In specificembodiments, said first oligonucleotide and said second oligonucleotideare joined in a head-to-head or tail-to-tail conformation. In specificembodiments, when said multimerizing or dimerizing oligonucleotide ofsaid multimerizing agent or dimerizing agent hybridizes to theartificial oligonucleotide sequence of two of said cell deathpolypeptides, the cell death polypeptides are multimerized or dimerized.In another specific embodiment, when the cell death polypeptides aremultimerized or dimerized, an apoptosis-inducing signal is generated insaid cell. In particular embodiments, the cell death polypeptidescomprise intracellular caspase domains, and when the intracellularcaspase domains are multimerized or dimerized, an apoptosis-inducingsignal is generated in said cell.

In certain other embodiments of the cell, e.g., T lymphocyte, saidmultimerizing or dimerizing agent is an artificial polypeptidecomprising two or more binding domains joined by one or more linkers.

In a specific embodiment, provided herein is a cell, e.g., a Tlymphocyte, comprising a cell death polypeptide comprising anextracellular domain comprising an epitope, a transmembrane domain, andan intracellular domain comprising a caspase 9, e.g., a human caspase 9,or a functional portion thereof. In another specific embodiment,provided herein is a cell, e.g., a T lymphocyte, comprising anartificial polypeptide comprising an extracellular domain comprising areceptor or ligand-binding portion thereof, and an intracellular domaincomprising a caspase 9, e.g., a human caspase 9, or a functional portionthereof. In another specific embodiment, provided herein is a cell,e.g., a T lymphocyte, comprising an artificial polypeptide comprising anextracellular domain comprising a ligand or a receptor-binding portionthereof, wherein said ligand binds a receptor or ligand-binding portionthereof, and an intracellular domain comprising a caspase 9, e.g., ahuman caspase 9, or functional portion thereof. In a specificembodiment, said cell is a T lymphocyte.

In another aspect, provided herein is cell, e.g., a T lymphocyte, safetysystem comprising a cell comprising (a) a cell death polypeptidecomprising an extracellular domain comprising an epitope, atransmembrane domain, and an intracellular domain comprising a caspaseor a functional portion thereof; and (b) a dimerizing agent comprisingtwo epitope-binding or mimotope-binding domains that when contacted withtwo of said cell death polypeptides dimerizes said polypeptides, whereinsaid caspase is caspase 3, caspase 8 or caspase 9, e.g., human caspase3, caspase 8, or caspase 9, and wherein said dimerization generates anapoptosis-inducing signal in said cell. In a specific embodiment, saidcell is a T lymphocyte.

In another embodiment, provided herein is a cell, e.g., a T lymphocyte,safety system comprising (a) a cell comprising an artificial polypeptidecomprising an extracellular domain comprising a receptor orligand-binding portion thereof, and an intracellular domain comprising acaspase or a functional portion thereof; and (b) a dimerizing agentcomprising two ligands that bind to said receptor or ligand-bindingportion thereof, wherein when said dimerizing agent is contacted withtwo of said polypeptides said dimerizing agent dimerizes saidpolypeptides, wherein said caspase is caspase 3, caspase 8 or caspase 9,e.g., human caspase 3, caspase 8, or caspase 9, and wherein saiddimerization generates an apoptosis-inducing signal in said cell. In aspecific embodiment, said cell is a T lymphocyte.

In another embodiment, provided herein is a cell, e.g., a T lymphocyte,safety system comprising (a) a cell comprising an artificial cell deathpolypeptide comprising an extracellular domain comprising a ligand or areceptor-binding portion thereof, and an intracellular domain comprisinga caspase or functional portion thereof; and (b) a dimerizing agentcomprising two receptors or ligand-binding portions thereof that bind tosaid ligand or receptor-binding portion thereof, wherein when saiddimerizing agent is contacted with two of said polypeptides saiddimerizing agent dimerizes said polypeptides, wherein said caspase iscaspase 3, caspase 8 or caspase 9, e.g., human caspase 3, caspase 8, orcaspase 9, and wherein said dimerization generates an apoptosis-inducingsignal in said cell. In a specific embodiment, said cell is a Tlymphocyte.

In a specific embodiment of any of the embodiments herein, when aplurality of said apoptosis-inducing signals are generated in said cell,e.g., T lymphocyte, said signal is sufficient to kill said cell. In aspecific embodiment, said cell is a T lymphocyte.

In another aspect, further provided herein is a method of killing acell, e.g., a T lymphocyte, wherein said cell comprises a plurality ofartificial cell death polypeptides each comprising an apoptosis-inducingdomain, wherein the cell death polypeptides are multimerizable ordimerizable using a multimerizing agent or dimerizing agent that is notan FK506 binding protein (FKBP) ligand, and wherein when saidmultimerizing agent or dimerizing agent multimerizes or dimerizes saidpolypeptide, an apoptosis-inducing signal is generated in said Tlymphocyte, comprising contacting said cell with an amount of saidmultimerizing agent or dimerizing agent sufficient for said plurality ofthe cell death polypeptides to multimerize or dimerize and generate anaggregate apoptosis-inducing signal sufficient to kill said cell. Incertain embodiments, the cell death polypeptide is a transmembranepolypeptide comprising an extracellular domain, a transmembrane domain,and an intracellular domain comprising said apoptosis-inducing domain.In specific embodiments, said apoptosis-inducing domain of saidpolypeptide is or comprises a caspase, e.g., caspase 3, caspase 8 orcaspase 9, for example a human caspase 9, caspase 8, or caspase 3. Inspecific embodiments, the multimerizing agent or dimerizing agent is aprotein, an oligonucleotide or a polysaccharide. In a specificembodiment, said cell is a T lymphocyte.

In certain embodiments, the multimerizing agent or dimerizing agent is aprotein, an oligonucleotide, or a polysaccharide. In specificembodiments, the multimerizing agent or dimerizing agent is apolypeptide comprising at least two sites that specifically bind to acell death polypeptide, e.g., an extracellular domain of a cell deathpolypeptide. In particular embodiments, the polypeptide is an antibody,e.g., an antibody that comprises at least two epitope-binding sites orat least two mimotope-binding sites. In certain embodiments, anextracellular domain of a cell death polypeptide comprises at least oneepitope or mimotope to which the antibody specifically binds. Inparticular embodiments, the antibody is a bispecific antibody comprisingtwo different epitope or mimotope binding sites that bind two differentepitopes or mimotopes present on an extracellular domain of a cell deathpolypeptide. In certain embodiments, the antibody is an IgG or an IgMantibody. In a specific embodiment, an antibody useful as amultimerizing or dimerizing agent is one that has been approved by theUnited States Food and Drug Administration for any use.

In a specific embodiment, when the multimerizing agent or dimerizingagent is an antibody, said antibody is one that specifically binds to aCD20 epitope or mimotope, e.g., a human CD20 epitope or mimotope, andsaid extracellular domain of a cell death polypeptide comprises a CD20epitope or mimotope to which said antibody specifically binds. Incertain specific embodiments, when the multimerizing agent or dimerizingagent is an antibody, said antibody is rituximab and said extracellulardomain of the cell death polypeptide comprises a CD20 epitope or a CD20mimotope that binds to said rituximab; said antibody is tositumumab andsaid extracellular domain of the cell death polypeptide comprises a CD20epitope or a CD20 mimotope that binds to said tositumumab; said antibodyis ibritumomab and said extracellular domain of the cell deathpolypeptide comprises a CD20 epitope or a CD20 mimotope that binds saidibritumomab; said antibody is ofatumumab and said extracellular domainof the cell death polypeptide comprises a CD20 epitope or a CD20mimotope that binds said ofatumumab; said antibody is alemtuzumab andsaid extracellular domain of the cell death polypeptide comprises a CD52epitope or a CD52 mimotope that binds to said alemtuzumab; said antibodyis basiliximab and said extracellular domain of the cell deathpolypeptide comprises a CD25 epitope or a CD25 mimotope that binds saidbasiliximab; said antibody is daclizumab and said extracellular domainof the cell death polypeptide comprises a CD25 epitope or a CD25mimotope that binds said daclizumab; said antibody is brentuximab andsaid extracellular domain of the cell death polypeptide comprises a CD30epitope or a CD30 mimotope that binds said brentuximab; said antibody isbelimumab and said extracellular domain of the cell death polypeptidecomprises a B-cell activating factor (BAFF) epitope or a BAFF mimotopethat binds said belimumab; said antibody is cetuximab and saidextracellular domain of the cell death polypeptide comprises anepidermal growth factor receptor (EGFR) epitope or an EGFR mimotope thatbinds said cetuximab; said antibody is panitumumab and saidextracellular domain of the cell death polypeptide comprises anepidermal growth factor receptor (EGFR) epitope or an EGFR mimotope thatbinds said panitumumab; said antibody is efalizumab and saidextracellular domain of the cell death polypeptide comprises an epitopeof CD11a or a mimotope of CD11a that binds to said efalizumab; saidantibody is ipilimumab and said extracellular domain of the cell deathpolypeptide comprises a CD152 epitope or CD152 mimotope that binds saidipilimumab; said antibody is natalizumab and said extracellular domainof the cell death polypeptide comprises an epitope of alpha-4 integrinor a mimotope of alpha 4 integrin that binds said natalizumab; or saidantibody is basiliximab and said extracellular domain of the cell deathpolypeptide comprises a CD25 epitope or CD25 mimotope that binds saidbasiliximab. In a specific embodiment of any of the above embodiments,when said antibody binds to said epitope or mimotope on at least two ofsaid cell death polypeptides, the intracellular domains of saidpolypeptides, and/or the respective caspases in said intracellulardomains multimerize or dimerize. In specific embodiments, when saidantibody specifically binds to an epitope or mimotope of at least twocell death polypeptides, dimerization of the cell death polypeptidesoccurs, e.g., dimerization of the intracellular domains of the celldeath polypeptides occurs. In particular embodiments the cell deathpolypeptides comprise intracellular domains comprising a caspase domain,and dimerization of the caspase domains occurs. In specific embodiments,said dimerization, for example, dimerization of intracellular domains,e.g., dimerization of caspase domains, initiates an apoptosis-inducingsignal in said cell, e.g., T lymphocyte.

In certain specific embodiments, said extracellular domain of the celldeath polypeptide is or comprises a receptor or a ligand-binding portionthereof. In such embodiments, said multimerizing agent or dimerizingagent comprises at least two ligands for said receptor or ligand bindingportion thereof. In specific embodiments, when said multimerizing agentor dimerizing agent binds to said receptor or said ligand bindingportion thereof on at least two of said cell death polypeptides, saidpolypeptides are multimerized or dimerized. In specific embodiments,said multimerization or dimerization of said polypeptides initiates anapoptosis-inducing signal in said cell, e.g., T lymphocyte.

In certain specific embodiments, said extracellular domain of the celldeath polypeptide comprises a ligand for a receptor. In suchembodiments, said multimerizing agent or dimerizing agent comprises atleast two receptors or ligand-binding portions thereof that bind to saidligand. In specific embodiments, when said multimerizing agent ordimerizing agent binds to said receptor or said ligand binding portionthereof on two or more of said polypeptides, the intracellular domainsin said polypeptides and/or the caspase domains of said polypeptides aremultimerized or dimerized. In specific embodiments, said multimerizationor dimerization of said intracellular domains and/or caspase domainsinitiates an apoptosis-inducing signal is generated in said cell, e.g.,T lymphocyte.

In certain embodiments, said extracellular domain of said cell deathpolypeptide comprises an artificial oligonucleotide sequence. Forexample, in particular embodiments, said cell death polypeptidecomprises an extracellular domain that comprises an artificialoligonucleotide sequence. In a specific embodiment, a multimerizing ordimerizing agent is or comprises at least one multimerizing ordimerizing oligonucleotide comprising a first oligonucleotide and asecond oligonucleotide, optionally joined by a linker, wherein saidfirst oligonucleotide and said second oligonucleotide are complementaryto said artificial oligonucleotide sequence. In certain specificembodiments, said first oligonucleotide and said second oligonucleotidehave the same sequence. In specific embodiments, said firstoligonucleotide and said second oligonucleotide are joined in ahead-to-head or tail-to-tail conformation. In specific embodiments, whensaid multimerizing or dimerizing oligonucleotide of said multimerizingagent or dimerizing agent hybridizes to the artificial oligonucleotidesequence of two of said cell death polypeptides, the cell deathpolypeptides are multimerized or dimerized. In another specificembodiment, when the cell death polypeptides are multimerized ordimerized, an apoptosis-inducing signal is generated in said cell. Inparticular embodiments, the cell death polypeptides compriseintracellular caspase domains, and when the intracellular caspasedomains are multimerized or dimerized, an apoptosis-inducing signal isgenerated in said cell. In a specific embodiment, said cell is a Tlymphocyte.

In a specific embodiment of any of the embodiments herein, when aplurality of said apoptosis-inducing signals are generated in said Tlymphocyte, said signal is sufficient to kill said cell, e.g., Tlymphocyte.

In other embodiments, said multimerizing agent or dimerizing agent is anartificial polypeptide comprising two or more binding domains joined byone or more linkers.

In a specific embodiment, provided herein is a method of killing a cell,e.g., a T lymphocyte, wherein said cell comprises a plurality ofartificial cell death polypeptides each comprising a caspase orfunctional portion thereof, wherein said caspase is caspase 3, caspase 8or caspase 9 (e.g., human caspase 3, caspase 8, or caspase 9), and,wherein said cell death polypeptide is dimerizable using an antibody,and wherein when said antibody dimerizes said polypeptide, anapoptosis-inducing signal is generated in said cell, comprisingcontacting said cell with an amount of said antibody sufficient todimerize a sufficient number of said plurality of artificialpolypeptides to dimerize and generate an aggregate apoptosis-inducingsignal sufficient to kill said cell. In a specific embodiment, said cellis a T lymphocyte.

In another specific embodiment, provided herein is a method of killing acell, e.g., a T lymphocyte, wherein said cell comprises a plurality ofartificial cell death polypeptides each comprising a caspase orfunctional portion thereof, wherein said caspase is caspase 3, caspase 8or caspase 9 (e.g., human caspase 3, caspase 8, or caspase 9), and,wherein said plurality of artificial polypeptides each comprises anextracellular domain comprising a receptor or ligand-binding portionthereof that bind a ligand, wherein said polypeptide is dimerizableusing a dimerizing agent comprising two said ligands, and wherein whensaid dimerizing agent dimerizes two of said polypeptides, anapoptosis-inducing signal is generated in cell, comprising contactingsaid cell with an amount of said dimerizing agent sufficient to dimerizea sufficient number of said plurality of artificial cell deathpolypeptides to dimerize and generate an aggregate apoptosis-inducingsignal sufficient to kill said cell. In a specific embodiment, said cellis a T lymphocyte.

In another specific embodiment, provided herein is a method of killing acell, e.g., a T lymphocyte, wherein said cell comprises a plurality ofartificial cell death polypeptides each comprising a caspase orfunctional portion thereof, wherein said caspase is caspase 3, caspase 8or caspase 9 (e.g., human caspase 3, caspase 8, or caspase 9), and,wherein said plurality of artificial polypeptides each comprises anextracellular domain comprising a ligand or receptor-binding portionthereof that bind a receptor or ligand-binding portion thereof, whereinsaid polypeptides are dimerizable using a dimerizing agent comprisingtwo said receptors or ligand-binding portion thereof, and wherein whensaid dimerizing agent dimerizes two of said polypeptides, anapoptosis-inducing signal is generated in said cell, comprisingcontacting said cell with an amount of said dimerizing agent sufficientto dimerize a sufficient number of said plurality of artificialpolypeptides to dimerize and generate an aggregate apoptosis-inducingsignal sufficient to kill said cell. In a specific embodiment, said cellis a T lymphocyte.

In another specific embodiment, provided herein is a method of killing acell, e.g., a T lymphocyte, wherein said cell comprises a plurality ofartificial cell death polypeptides each comprising a caspase orfunctional portion thereof, wherein said caspase is caspase 3, caspase 8or caspase 9 (e.g., human caspase 3, caspase 8, or caspase 9), and,wherein said plurality of artificial polypeptides each comprising anextracellular domain comprising an artificial oligonucleotide, whereinsaid plurality of polypeptides are dimerizable using a dimerizing agentcomprising an oligonucleotide comprising a first oligonucleotide and asecond oligonucleotide, wherein said first oligonucleotide and saidsecond oligonucleotide have the same nucleotide sequence, and whereinsaid first oligonucleotide and second oligonucleotide optionally arejoined by a linker, and wherein said first oligonucleotide and saidsecond oligonucleotide are complementary to said artificialoligonucleotide in said extracellular domain of said polypeptide, andwherein when said dimerizing agent dimerizes two of said cell deathpolypeptides, an apoptosis-inducing signal is generated in said cell,comprising contacting said cell with an amount of said dimerizing agentsufficient to dimerize a sufficient number of said plurality ofartificial polypeptides to dimerize and generate an aggregateapoptosis-inducing signal sufficient to kill said cell. In a specificembodiment, said cell is a T lymphocyte.

In certain embodiments, the cells (e.g., T lymphocytes) killed inaccordance with the methods described herein comprise a polypeptide thatacts to target the cell to a particular antigen, e.g., atumor-associated antigen or tumor-specific antigen, wherein saidpolypeptide, when bound to said antigen, causes the cell to kill a celldisplaying said antigen, for example, a chimeric antigen receptor (CAR).T lymphocytes comprising CARs are referred to herein as CAR-Tlymphocytes. The chimeric antigen receptors typically comprise (i) anintracellular domain (e.g., cytoplasmic domain) of an endogenous proteinexpressed on the surface of lymphocytes and that triggers the activationand/or proliferation of said lymphocytes, (ii) a transmembrane domain,and (iii) an extracellular domain that binds to an antigen of interest,e.g., a tumor-associated antigen or tumor-specific antigen. The CAR-Tlymphocytes also typically comprise one or more co-stimulatory domains.In certain embodiments, a CAR-T lymphocyte comprises at least two CARpolypeptides, at least one of which provides a primary stimulatorysignal to the CAR-T lymphocyte, and at least one that provides acostimulatory signal to the CAR-T lymphocyte. CAR-T lymphocytescomprising a cell death polypeptide and comprising specific embodimentsof CARs are provided below.

In another aspect, provided herein are methods of treating an individualhaving a disease or disorder, wherein the disease or disorder ischaracterized, or is characterizable, by cells expressing an antigen,comprising administering to the individual cells, e.g., T lymphocytes,expressing a polypeptide, as described herein. In certain embodiments,when the modified cells, e.g., modified T lymphocytes described hereinare administered to a subject in need thereof, the combination ofmultimerizing agent and cell death polypeptide selected are chosen suchthat they are compatible with the patient population (or subpopulation)in which the cells, e.g., T lymphocytes, have been administered. By wayof example only, if the multimerizing agent selected is the antibodyrituximab, then in certain embodiments the patient population isindividuals having a cancer of the B cells, e.g., B cell lymphoma.

4. DETAILED DESCRIPTION 4.1. Cells Comprising Cell Death Polypeptides

Provided herein are genetically modified cells, for example immunecells, such as T lymphocytes, e.g., human T lymphocytes, that comprisean artificial multimerizable, e.g., dimerizable, polypeptide (referredto herein as a “cell death polypeptide”) that, when multimerized, e.g.,dimerized, by a multimerizing agent, e.g., dimerizing agent, generatesan apoptosis-inducing signal in a cell, e.g., a T lymphocyte, thatexpresses the polypeptide, resulting in cell death, e.g., via apoptosis.Without wishing to be bound by any particular mechanism or theory, it isthought that when a sufficient number of a plurality of cell deathpolypeptides of the cell are multimerized, e.g., dimerized, that theaggregate apoptosis-inducing signal thereby generated is sufficient tokill the cell, e.g., cause the cell to undergo apoptosis. In a specificembodiment, the genetically modified cells provided herein are Tlymphocytes.

In certain embodiments, the cell death polypeptide can be multimerizedor dimerized by an administrable multimerizing agent or dimerizingagent, e.g., a protein (e.g., antibody, receptor or ligand-bindingportion thereof, a ligand or receptor-binding portion thereof),oligonucleotide, or the like. In certain embodiments, the multimerizingagent is not a small molecule. The multimerizing or dimerizing agent canbe used to kill T lymphocytes comprising the cell death polypeptideeither in vitro or in vivo.

Thus, in a first aspect, provided herein is a T lymphocyte comprising anartificial polypeptide (cell death polypeptide) comprising anapoptosis-inducing domain, wherein said cell death polypeptide ismultimerizable using a multimerizing agent, wherein said multimerizingagent is not an FK506 binding protein (FKBP) ligand, and wherein whensaid multimerizing agent multimerizes said polypeptide, anapoptosis-inducing signal is generated in said T lymphocyte. In aspecific embodiment, said multimerizing agent is a dimerizing agent;that is, the multimerizing agent causes the cell death polypeptide todimerize. In another specific embodiment, when said dimerizing agentdimerizes said cell death polypeptide, an apoptosis-inducing signal isgenerated in said T lymphocyte. The cell death polypeptide does notcomprise an FK506 binding protein, functional portion thereof, ormodified form thereof.

In certain embodiments, said cell death polypeptide is a transmembranepolypeptide comprising an extracellular domain, a transmembrane domain,and an intracellular domain comprising said apoptosis-inducing domain.In certain embodiments, the apoptosis-inducing domain of the cell deathpolypeptide can be, for example, any protein or portion thereof thatwhen dimerized initiates an apoptosis-inducing signal in the cell. Incertain embodiments, the apoptosis-inducing domain is any caspase thathomodimerizes, and preferably is or comprises a caspase, e.g., caspase9, caspase 8, or caspase 3 (e.g., human caspase 9, caspase 8, or caspase3). The amino acid sequences of human caspases, including human caspase9, human caspase 8, and human caspase 3 are well known in the art. Forexample, human caspase 3 has been assigned NCBI Gene ID: 836; humancaspase 8 has been assigned NCBI Gene ID: 841; and human caspase 9 hasbeen assigned NCBI Gene ID: 842. In certain embodiments, theintracellular domain that is, or comprises, a caspase domain, and theextracellular domain, which comprises the epitope or mimotope, arejoined by a CD8α stalk or CD8β stalk, at least part of which canfunction as a transmembrane domain.

In certain embodiments, the dimerizing agent is a polypeptide comprisingat least two sites that specifically bind to a cell death polypeptide,e.g., an extracellular domain of a cell death polypeptide. In particularembodiments, the polypeptide is an antibody, e.g., an antibodycomprising at least two epitope or mimotope binding sites. In certainembodiments, only the antigen binding domain of an antibody is used as amultimerizing or dimerizing agent. In certain embodiments, anextracellular domain of a cell death polypeptide comprises at least oneepitope or mimotope to which the antibody specifically binds. Inparticular embodiments, the antibody is a bispecific antibody comprisingtwo different epitope or mimotope binding sites that bind two differentepitopes or mimotopes present on an extracellular domain of a cell deathpolypeptide. In certain embodiments, the antibody is an IgG or an IgMantibody. Artificial antibody constructs comprising epitope-binding ormimotope-binding domains from antibodies, optionally joined by one ormore linkers, may also be used.

In a specific embodiment, said antibody, useful as a multimerizing ordimerizing agent, has been approved by a governmental regulatoryauthority, e.g., the United States Food and Drug Administration for anyuse. This ensures, e.g., that the antibody, when used as a dimerizing ormultimerizing agent, has a known toxicity and patient safety profile.Any combination of antibody and associated target may be used in the Tlymphocytes provided herein. In one embodiment, an antibody useful as amultimerizing or dimerizing agent is one that specifically binds to aCD20 epitope or mimotope, e.g., a human CD20 epitope or mimotope, and anextracellular domain of a cell death polypeptide comprises a CD20epitope or mimotope to which the antibody specifically binds. In certainspecific embodiments, the antibody is rituximab and an extracellulardomain of a cell death polypeptide comprises a CD20 epitope or a CD20mimotope that specifically binds to said rituximab. In another specificembodiment, the antibody is tositumumab and an extracellular domain of acell death polypeptide comprises a CD20 epitope or a CD20 mimotope thatspecifically binds to said tositumumab. In yet another embodiment, theantibody is ibritumomab and an extracellular domain of a cell deathpolypeptide comprises a CD20 epitope or a CD20 mimotope thatspecifically binds to said ibritumomab. In still another embodiment, theantibody is ofatumumab and an extracellular domain of a cell deathpolypeptide comprises a CD20 epitope or a CD20 mimotope thatspecifically binds to said atumumab.

In another specific embodiment, the antibody is alemtuzumab and anextracellular domain of a cell death polypeptide comprises a CD52epitope or a CD52 mimotope that specifically binds to said alemtuzumab.In yet another embodiment, the antibody is basiliximab and anextracellular domain of the cell death polypeptide comprises a CD25epitope or a CD25 mimotope that specifically binds to said basiliximab.In another embodiment, the antibody is daclizumab and an extracellulardomain of a cell death polypeptide comprises a CD25 epitope or a CD25mimotope that specifically binds to said daclizumab. In still anotherembodiment, the antibody is brentuximab and an extracellular domain of acell death polypeptide comprises a CD30 epitope or a CD30 mimotope thatspecifically binds to said brentuximab. In another embodiment, theantibody is belimumab and an extracellular domain of a cell deathpolypeptide comprises a B-cell activating factor (BAFF) epitope or aBAFF mimotope that specifically binds to said belimumab. In anotherembodiment, the antibody is cetuximab and an extracellular domain of acell death polypeptide comprises an epidermal growth factor receptor(EGFR) epitope or an EGFR mimotope that specifically binds to saidcetuximab. In yet another embodiment, the antibody is panitumumab and anextracellular domain of a cell death polypeptide comprises an epidermalgrowth factor receptor (EGFR) epitope or an EGFR mimotope thatspecifically binds to said panitumumab. In another embodiment, theantibody is efalizumab and an extracellular domain of a cell deathpolypeptide comprises an epitope of CD11a or a mimotope of CD11a thatspecifically binds to said efalizumab. In still another embodiment, theantibody is ipilimumab and an extracellular domain of a cell deathpolypeptide comprises a CD152 epitope or CD152 mimotope thatspecifically binds to said ipilimumab. In still another embodiment, theantibody is natalizumab and an extracellular domain of a cell deathpolypeptide comprises an epitope of alpha-4 integrin or a mimotope ofalpha 4 integrin that specifically binds to said natalizumab. In anotherembodiment, the antibody is basiliximab and an extracellular domain of acell death polypeptide comprises a CD25 epitope or CD25 mimotope thatspecifically binds to said basiliximab.

Ligands and receptors can be utilized in the construction of the celldeath polypeptides provided herein, and multimerizing agents ordimerizing agents comprising the receptors' respective ligands can beused to multimerize or dimerize the polypeptides. In certainembodiments, when a multimerizing agent or a dimerizing agent binds toat least two cell death polypeptides, dimerization or multimerization ofthe cell death polypeptides occurs, e.g., dimerization ormultimerization of the cell death polypeptides occurs. In certainembodiments, an extracellular domain of a cell death polypeptide is orcomprises a receptor or a ligand-binding portion thereof. In a specificembodiment, a multimerizing agent or dimerizing agent is or comprises atleast two ligands for said receptor or ligand binding portion thereof.In another specific embodiment, said multimerizing agent or dimerizingagent binds to said receptor or said ligand binding portion thereof ontwo of the cell death polypeptides, and said polypeptides aremultimerized or dimerized, e.g., the intracellular domains of saidpolypeptides are multimerized or dimerized. In particular embodimentsthe cell death polypeptides comprise intracellular domains comprising acaspase domain, and multimerization or dimerization of the caspasedomains occurs. In specific embodiments, said multimerization ordimerization, for example, multimerization or dimerization ofintracellular domains, e.g., multimerization or dimerization of caspasedomains, initiates an apoptosis-inducing signal in said cell, e.g., Tlymphocyte.

In specific embodiments, when an antibody specifically binds to anepitope or mimotope of at least two cell death polypeptides,dimerization of the cell death polypeptides occurs, e.g., dimerizationof the intracellular domains of the cell death polypeptides occurs. Inparticular embodiments the cell death polypeptides compriseintracellular domains comprising a caspase domain, and dimerization ofthe caspase domains occurs. In specific embodiments, said dimerization,for example, dimerization of intracellular domains, e.g., dimerizationof caspase domains, initiates an apoptosis-inducing signal in said cell,e.g., T lymphocyte.

In certain other embodiments of the cell, e.g., T lymphocyte, saidextracellular domain of the cell death polypeptide comprises a ligandfor a receptor. In a specific embodiment, said multimerizing agent ordimerizing agent comprises at least two receptors or ligand-bindingportions thereof that bind to said ligand. In a specific embodiment,when said multimerizing agent or dimerizing agent binds to said receptoror said ligand binding portion thereof on at least two of the cell deathpolypeptides, said polypeptides are multimerized or dimerized. In aspecific embodiment, when the cell death polypeptides are multimerizedor dimerized, an apoptosis-inducing signal is generated in said cell. Ina specific embodiment, said cell is a T lymphocyte.

In certain other embodiments, an extracellular domain of a cell deathpolypeptide comprises an artificial oligonucleotide sequence. Forexample, in particular embodiments, a modified cell, e.g., T lymphocyte,comprises a cell death polypeptide comprising an extracellular domainthat comprises an artificial oligonucleotide sequence. In a specificembodiment, a multimerizing or dimerizing agent is or comprises at leastone multimerizing or dimerizing oligonucleotide comprising a firstoligonucleotide and a second oligonucleotide, optionally joined by alinker, wherein said first oligonucleotide and said secondoligonucleotide are complementary to said artificial oligonucleotidesequence. In certain specific embodiments, said first oligonucleotideand said second oligonucleotide have the same sequence. In specificembodiments, said first oligonucleotide and said second oligonucleotideare joined in a head-to-head or tail-to-tail conformation. In specificembodiments, when said multimerizing or dimerizing oligonucleotide ofsaid multimerizing agent or dimerizing agent hybridizes to theartificial oligonucleotide sequence of two of said cell deathpolypeptides, the cell death polypeptides are multimerized or dimerized.In another specific embodiment, when the cell death polypeptides aremultimerized or dimerized, an apoptosis-inducing signal is generated insaid cell. In particular embodiments, the cell death polypeptidescomprise intracellular caspase domains, and when the intracellularcaspase domains are multimerized or dimerized, an apoptosis-inducingsignal is generated in said cell. In a specific embodiment, said cell isa T lymphocyte.

In certain other embodiments of the T lymphocyte, the multimerizing ordimerizing agent is an artificial polypeptide comprising two or morebinding domains joined by one or more linkers.

In a specific embodiment, provided herein is a cell, e.g., a Tlymphocyte, comprising a cell death polypeptide comprising anextracellular domain comprising an epitope, a transmembrane domain, andan intracellular domain comprising a caspase 9, e.g., a human caspase 9,or a functional portion thereof. In another specific embodiment,provided herein is a cell, e.g., a T lymphocyte, comprising anartificial polypeptide comprising an extracellular domain comprising areceptor or ligand-binding portion thereof, and an intracellular domaincomprising a caspase 9, e.g., a human caspase 9, or a functional portionthereof. In another specific embodiment, provided herein is a cell,e.g., a T lymphocyte, comprising an artificial polypeptide comprising anextracellular domain comprising a ligand or a receptor-binding portionthereof, wherein said ligand binds a receptor or ligand-binding portionthereof, and an intracellular domain comprising a caspase 9, e.g., ahuman caspase 9, or functional portion thereof. In a specificembodiment, said cell is a T lymphocyte.

In any of the embodiments herein, wherein the modified cells are Tlymphocytes, the T lymphocytes may be CD4+ T lymphocytes or CD8+ Tlymphocytes. The T lymphocytes may be, without genetic modification,specific for a particular antigen (e.g., a tumor-associated antigen,tumor-specific antigen, viral antigen, or the like). The T lymphocytesmay be genetically modified to express one or more polypeptides, e.g.,chimeric antigen receptors, that target the T lymphocyte to a specificantigen.

4.2. Methods of Killing Cells that Comprise Cell Death Polypeptides

The cell death polypeptides provided herein can be used in methods ofkilling cells, e.g., T lymphocytes, that comprise the cell deathpolypeptides. The cell death polypeptides provided herein may be used inconjunction with any cells, in particular, any mammalian cells, forexample, any human cells. Such cell death polypeptides provide, forexample, a useful safety feature for cell therapeutics. As such, thecell death polypeptides can, for example, be important for a drugproduct comprising a cell therapeutic, e.g., a chimeric antigenreceptor-expressing CAR T lymphocytes, because the cell deathpolypeptides enable rapid killing of the cell therapeutic, e.g., the Tlymphocytes, should such rapid killing become desirable, e.g., in theevent administration of the cells causes any unwanted or deleteriouseffects in a patient receiving them, or if the presence of the celltherapeutic, e.g., the T lymphocytes, in a subject is no longernecessary. Thus, in certain embodiments, the cell death polypeptidesprovided herein can be used in conjunction with any administrable cells,for example cell therapeutics, such as mammalian cell therapeutics,e.g., human cell therapeutics. Non-limiting examples of cells in whichthe cell death polypeptides and multimerizing or dimerizing agents maybe used include, but are not limited to, natural killer (NK) cells,dendritic cells (DC), placental stem cells (e.g., the placental stemcells disclosed in U.S. Pat. Nos. 7,468,276; 8,057,788 and 8,202,703,the disclosures of which are hereby incorporated by reference in theirentireties), mesenchymal-like stem cells from umbilical cord blood,placental blood, peripheral blood, bone marrow, dental pulp, adiposetissue, osteochondral tissue, and the like; embryonic stem cells,embryonic germ cells, neural crest stem cells, neural stem cells, anddifferentiated cells (e.g., fibroblasts, etc.). The cell deathpolypeptides, and multimerizing or dimerizing agents, may also be usedin tumor cell lines, e.g., for animal model experimental purposes.

Cell killing by the cell death polypeptides described herein can takeplace either in vivo, e.g., in an individual to whom the cells, e.g., Tlymphocytes, have been administered, or in vitro, e.g., in a laboratory,e.g., as part of quality control experiments. In one embodiment,provided herein is a method of killing a cell, e.g., a T lymphocyte,wherein said cell comprises a plurality of artificial cell deathpolypeptides each comprising an apoptosis-inducing domain, wherein saidpolypeptides are multimerizable or dimerizable using a multimerizingagent or dimerizing agent that is not an FK506 binding protein (FKBP)ligand, and wherein when said multimerizing agent or dimerizing agentmultimerizes or dimerizes said polypeptide, an apoptosis-inducing signalis generated in said cell, comprising contacting said cell with anamount of said multimerizing agent or dimerizing agent sufficient forsaid plurality of artificial polypeptides to dimerize and generate anaggregate apoptosis-inducing signal sufficient to kill said cell. Incertain embodiments, the cell death polypeptide is a transmembranepolypeptide comprising an extracellular domain, a transmembrane domain,and an intracellular domain comprising said apoptosis-inducing domain.In certain embodiments, the intracellular domain that is, or comprises,a caspase domain, and the extracellular domain, which comprises theepitope or mimotope, are joined by a CD8α stalk or CD8β stalk, at leastpart of which can function as a transmembrane domain. In certainspecific embodiments of the method, the apoptosis-inducing domain ofsaid polypeptide is or comprises a caspase, e.g., caspase 3, caspase 8,or caspase 9 (e.g., human caspase 9, caspase 8, or caspase 3).

The multimerizing agent or dimerizing agent used in the method can beany compound, other than a small molecule, that can dimerize ormultimerizes a cell death polypeptide, e.g., a protein, anoligonucleotide or a polysaccharide. In certain embodiments, themultimerizing agent or dimerizing agent is an antibody, e.g., anantibody that comprises at least two epitope-binding sites or at leasttwo mimotope-binding sites. In certain embodiments, only the antigenbinding domain of an antibody is used as a multimerizing or dimerizingagent. In such embodiments, the extracellular domain of the cell deathpolypeptide comprises an epitope or mimotope to which the antibodybinds. The antibody can be an antibody of any valence, but is preferablyan IgG or an IgM antibody.

In a specific embodiment, said antibody, useful as a multimerizing ordimerizing agent, has been approved by a governmental regulatoryauthority, e.g., the United States Food and Drug Administration for anyuse. Any combination of antibody and associated target may be used inthe methods of killing T lymphocytes provided herein.

In a specific embodiment, when the multimerizing agent or dimerizingagent is an antibody, said antibody is one that specifically binds to aCD20 epitope or mimotope, e.g., a human CD20 epitope or mimotope, andsaid extracellular domain of a cell death polypeptide comprises a CD20epitope or mimotope to which said antibody specifically binds. Incertain specific embodiments, when the multimerizing agent or dimerizingagent is an antibody, said antibody is rituximab and said extracellulardomain of the cell death polypeptide comprises a CD20 epitope or a CD20mimotope that binds to said rituximab; said antibody is tositumumab andsaid extracellular domain of the cell death polypeptide comprises a CD20epitope or a CD20 mimotope that binds to said tositumumab; said antibodyis ibritumomab and said extracellular domain of the cell deathpolypeptide comprises a CD20 epitope or a CD20 mimotope that binds saidibritumomab; said antibody is ofatumumab and said extracellular domainof the cell death polypeptide comprises a CD20 epitope or a CD20mimotope that binds said ofatumumab; or said antibody is alemtuzumab andsaid extracellular domain of the cell death polypeptide comprises a CD52epitope or a CD52 mimotope that binds to said alemtuzumab.

In certain specific embodiments, when the multimerizing agent ordimerizing agent is an antibody, said antibody is said antibody isbasiliximab and said extracellular domain of the cell death polypeptidecomprises a CD25 epitope or a CD25 mimotope that binds said basiliximab;said antibody is daclizumab and said extracellular domain of the celldeath polypeptide comprises a CD25 epitope or a CD25 mimotope that bindssaid daclizumab; said antibody is brentuximab and said extracellulardomain of the cell death polypeptide comprises a CD30 epitope or a CD30mimotope that binds said brentuximab; said antibody is belimumab andsaid extracellular domain of the cell death polypeptide comprises aB-cell activating factor (BAFF) epitope or a BAFF mimotope that bindssaid belimumab; said antibody is cetuximab and said extracellular domainof the cell death polypeptide comprises an epidermal growth factorreceptor (EGFR) epitope or an EGFR mimotope that binds said cetuximab;said antibody is panitumumab and said extracellular domain of the celldeath polypeptide comprises an epidermal growth factor receptor (EGFR)epitope or an EGFR mimotope that binds said panitumumab; said antibodyis efalizumab and said extracellular domain of the cell deathpolypeptide comprises an epitope of CD11a or a mimotope of CD11a thatbinds to said cfalizumab; said antibody is ipilimumab and saidextracellular domain of the cell death polypeptide comprises a CD152epitope or CD152 mimotope that binds said ipilimumab; said antibody isnatalizumab and said extracellular domain of the cell death polypeptidecomprises an epitope of alpha-4 integrin or a mimotope of alpha 4integrin that binds said natalizumab; or said antibody is basiliximaband said extracellular domain of the cell death polypeptide comprises aCD25 epitope or CD25 mimotope that binds said basiliximab.

In a specific embodiment of any of the above embodiments, when saidantibody binds to said epitope or mimotope on at least two of said celldeath polypeptides, the intracellular domains of said polypeptides,and/or the respective caspases in said intracellular domains multimerizeor dimerize. In specific embodiments, when said antibody specificallybinds to an epitope or mimotope of at least two cell death polypeptides,dimerization of the cell death polypeptides occurs, e.g., dimerizationof the intracellular domains of the cell death polypeptides occurs. Inparticular embodiments the cell death polypeptides compriseintracellular domains comprising a caspase domain, and dimerization ofthe caspase domains occurs. In specific embodiments, said dimerization,for example, dimerization of intracellular domains, e.g., dimerizationof caspase domains, initiates an apoptosis-inducing signal in said cell,e.g., T lymphocyte.

Without intending to be limited by theory, when the antibody binds tothe respective epitopes or mimotopes on at least two of saidpolypeptides, the intracellular domains of said polypeptidesmultimerizes, e.g., dimerize, at which time the respective caspases insaid intracellular domains dimerize. Dimerization of said polypeptidesinitiates an apoptosis-inducing signal in said T lymphocyte.

As above, receptors and their respective ligands may be used tomultimerize or dimerize cell death polypeptides, and thereby effectkilling of a cell, e.g., a T lymphocyte, comprising the polypeptide. Forexample, the extracellular domain of said cell death polypeptide is orcomprises a receptor or a ligand-binding portion thereof. In suchembodiments, the multimerizing agent or dimerizing agent comprises atleast two ligands for said receptor or ligand binding portion thereof,enabling multimerization or dimerization of the cell death polypeptidewhen the multimerizing agent or dimerizing agent binds to said receptoror said ligand binding portion thereof on at least two of saidpolypeptides, said polypeptides are dimerized. In preferred embodiments,dimerization of said polypeptides initiates an apoptosis-inducing signalin said cell.

In other embodiments of the method of killing cells, e.g., Tlymphocytes, the extracellular domain of the cell death polypeptidecomprises a ligand for a receptor. In such embodiments of the method,the multimerizing agent or dimerizing agent comprises at least tworeceptors or ligand-binding portions thereof that bind to said ligand.When the multimerizing dimerizing agent binds to said receptor or saidligand binding portion thereof on two of said cell death polypeptides,the intracellular domains, and thus preferably the caspase domains, insaid polypeptides are dimerized. Dimerization of said intracellulardomains, and the caspase domains, preferably initiates anapoptosis-inducing signal is generated in said cell.

In certain other embodiments of the method of killing cells, e.g., Tlymphocytes, said extracellular domain of said cell death polypeptidecomprises an artificial oligonucleotide sequence. For example, inparticular embodiments, said cell death polypeptide comprises anextracellular domain that comprises an artificial oligonucleotidesequence. In a specific embodiment, a multimerizing or dimerizing agentis or comprises at least one multimerizing or dimerizing oligonucleotidecomprising a first oligonucleotide and a second oligonucleotide,optionally joined by a linker, wherein said first oligonucleotide andsaid second oligonucleotide are complementary to said artificialoligonucleotide sequence. In certain specific embodiments, said firstoligonucleotide and said second oligonucleotide have the same sequence.In specific embodiments, said first oligonucleotide and said secondoligonucleotide are joined in a head-to-head or tail-to-tailconformation. In specific embodiments, when said multimerizing ordimerizing oligonucleotide of said multimerizing agent or dimerizingagent hybridizes to the artificial oligonucleotide sequence of two ofsaid cell death polypeptides, the cell death polypeptides aremultimerized or dimerized. In another specific embodiment, when the celldeath polypeptides are multimerized or dimerized, an apoptosis-inducingsignal is generated in said cell. In particular embodiments, the celldeath polypeptides comprise intracellular caspase domains, and when theintracellular caspase domains are multimerized or dimerized, anapoptosis-inducing signal is generated in said cell. In a specificembodiment, said cell is a T lymphocyte.

In certain other embodiments of the method of killing T lymphocytes, themultimerizing or dimerizing agent is an artificial polypeptidecomprising two or more binding domains joined by one or more linkers.

In a specific embodiment, provided herein is a method of killing a cell,e.g., a T lymphocyte, wherein said cell comprises a plurality ofartificial cell death polypeptides each comprising a caspase orfunctional portion thereof, wherein said caspase is caspase 3, caspase 8or caspase 9 (e.g., human caspase 3, caspase 8, or caspase 9), and,wherein said cell death polypeptide is dimerizable using an antibody,and wherein when said antibody dimerizes said polypeptide, anapoptosis-inducing signal is generated in said cell, comprisingcontacting said cell with an amount of said antibody sufficient todimerize a sufficient number of said plurality of artificialpolypeptides to dimerize and generate an aggregate apoptosis-inducingsignal sufficient to kill said cell. In a specific embodiment, said cellis a T lymphocyte.

In another specific embodiment, provided herein is a method of killing acell, e.g., a T lymphocyte, wherein said cell comprises a plurality ofartificial cell death polypeptides each comprising a caspase orfunctional portion thereof, wherein said caspase is caspase 3, caspase 8or caspase 9 (e.g., human caspase 3, caspase 8, or caspase 9), and,wherein said plurality of artificial polypeptides each comprises anextracellular domain comprising a receptor or ligand-binding portionthereof that bind a ligand, wherein said polypeptide is dimerizableusing a dimerizing agent comprising two said ligands, and wherein whensaid dimerizing agent dimerizes two of said polypeptides, anapoptosis-inducing signal is generated in cell, comprising contactingsaid cell with an amount of said dimerizing agent sufficient to dimerizea sufficient number of said plurality of artificial cell deathpolypeptides to dimerize and generate an aggregate apoptosis-inducingsignal sufficient to kill said cell. In a specific embodiment, said cellis a T lymphocyte.

In another specific embodiment, provided herein is a method of killing acell, e.g., a T lymphocyte, wherein said cell comprises a plurality ofartificial cell death polypeptides each comprising a caspase orfunctional portion thereof, wherein said caspase is caspase 3, caspase 8or caspase 9 (e.g., human caspase 3, caspase 8, or caspase 9), and,wherein said plurality of artificial polypeptides each comprises anextracellular domain comprising a ligand or receptor-binding portionthereof that bind a receptor or ligand-binding portion thereof, whereinsaid polypeptides are dimerizable using a dimerizing agent comprisingtwo said receptors or ligand-binding portion thereof, and wherein whensaid dimerizing agent dimerizes two of said polypeptides, anapoptosis-inducing signal is generated in said cell, comprisingcontacting said cell with an amount of said dimerizing agent sufficientto dimerize a sufficient number of said plurality of artificialpolypeptides to dimerize and generate an aggregate apoptosis-inducingsignal sufficient to kill said cell. In a specific embodiment, said cellis a T lymphocyte.

In another specific embodiment, provided herein is method of killing acell, e.g., a T lymphocyte, wherein said cell comprises a plurality ofartificial cell death polypeptides each comprising a caspase orfunctional portion thereof, wherein said caspase is caspase 3, caspase 8or caspase 9 (e.g., human caspase 3, caspase 8, or caspase 9), and,wherein said plurality of artificial polypeptides each comprising anextracellular domain comprising an artificial oligonucleotide, whereinsaid plurality of polypeptides are dimerizable using a dimerizing agentcomprising an oligonucleotide comprising a first oligonucleotide and asecond oligonucleotide, wherein said first oligonucleotide and saidsecond oligonucleotide have the same nucleotide sequence, and whereinsaid first oligonucleotide and second oligonucleotide optionally arejoined by a linker, and wherein said first oligonucleotide and saidsecond oligonucleotide are complementary to said artificialoligonucleotide in said extracellular domain of said polypeptide, andwherein when said dimerizing agent dimerizes two of said cell deathpolypeptides, an apoptosis-inducing signal is generated in said cell,comprising contacting said cell with an amount of said dimerizing agentsufficient to dimerize a sufficient number of said plurality ofartificial polypeptides to dimerize and generate an aggregateapoptosis-inducing signal sufficient to kill said cell. In a specificembodiment, said cell is a T lymphocyte.

In a specific embodiment, the T lymphocytes killed in accordance withthe methods described herein are CAR-T lymphocytes.

4.3. Chimeric Antigen Receptors

When the cells provided herein are T lymphocytes which comprise the celldeath polypeptides described above, such T lymphocytes can, in certainembodiments, comprise chimeric antigen receptors (CARs), which areartificial membrane-bound proteins that direct a T lymphocyte to anantigen, and stimulate the T lymphocyte to kill a cell displaying theantigen. See, e.g., Eshhar, U.S. Pat. No. 7,741,465. At a minimum, theCAR comprises an extracellular domain that binds to an antigen, e.g., anantigen on a cell, a transmembrane domain, and an intracellular(cytoplasmic) signaling domain that transmits a primary activationsignal to an immune cell. All other conditions being satisfied, when theCAR is expressed on the surface of, e.g., a T lymphocyte, and theextracellular domain of the CAR binds to an antigen, the intracellularsignaling domain transmits a signal to the T lymphocyte to activateand/or proliferate, and, if the antigen is present on a cell surface, tokill the cell expressing the antigen. Because T lymphocytes require twosignals, a primary activation signal and a costimulatory signal, inorder to activate, typically CARs also comprise a costimulatory domainsuch that binding of the antigen to the extracellular domain results intransmission of both a primary activation signal and a costimulatorysignal.

4.3.1. General CAR Structure Intracellular Domain

In certain embodiments, the intracellular domain of the CAR is orcomprises an intracellular domain or motif of a protein that isexpressed on the surface of T lymphocytes and triggers activation and/orproliferation of said T lymphocytes. Such a domain or motif is able totransmit a primary antigen-binding signal that is necessary for theactivation of a T lymphocyte in response to the antigen's binding to theCAR's extracellular portion. Typically, this domain or motif comprises,or is, an ITAM (immunoreceptor tyrosine-based activation motif).ITAM-containing polypeptides suitable for CARs include, for example, thezeta CD3 chain (CD3ζ) or ITAM-containing portions thereof. In a specificembodiment, the intracellular domain is a CD3ζ intracellular signalingdomain. In other specific embodiments, the intracellular domain is froma lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptorsubunit or an IL-2 receptor subunit.

In certain embodiments, the CAR additionally comprises one or moreco-stimulatory domains or motifs, e.g., as part of the intracellulardomain of the polypeptide. The one or more co-stimulatory domains ormotifs can be, or comprise, one or more of a co-stimulatory CD27polypeptide sequence, a co-stimulatory CD28 polypeptide sequence, aco-stimulatory OX40 (CD134) polypeptide sequence, a co-stimulatory 4-1BB(CD137) polypeptide sequence, or a costimulatory inducible T-cellcostimulatory (ICOS) polypeptide sequence, or other costimulatory domainor motif.

The transmembrane region can be any transmembrane region that can beincorporated into a functional CAR, typically a transmembrane regionfrom a CD4 or a CD8 molecule.

4.3.2. CAR Transmembrane Domains From CTLA4 or PD-1

In certain embodiments, the transmembrane domain of the CAR is from animmune system protein that normally transmits an inhibitory signal tosuch immune system cells, e.g., a transmembrane domain from CTLA4(Cytotoxic T-Lymphocyte Antigen 4 or Cytotoxic T-Lymphocyte Associatedprotein 4) or PD-1 (Programmed Death-1).

In certain embodiments, any of the T lymphocytes provided herein, whichcomprise a plurality of cell death polypeptides, comprise atransmembrane domain from CTLA4 or PD-1 (Programmed Cell Death 1) In aspecific embodiment, a T lymphocyte expressing said polypeptide, or anyof such polypeptides described herein, is activated or stimulated toproliferate when said polypeptide binds to said antigen. In a specificembodiment, the polypeptide, when expressed on the surface of a Tlymphocyte, directs the T lymphocyte to kill a cell expressing saidantigen.

In specific embodiments of any of the polypeptides herein, in which thetransmembrane domain of the polypeptide is from CTLA4, the CTLA4transmembrane domain is from a mammalian CTLA4, e.g., human, primate, orrodent, e.g., murine CTLA4. Preferably, the transmembrane domain doesnot comprise amino acids from the intracellular domain, extracellulardomain, or either intracellular or extracellular domain of CTLA4 orPD-1. Specific, non-limiting examples of CTLA4 or PD-1 transmembranedomain sequences are provided below.

In a specific embodiment, the CTLA4 transmembrane domain is thepolypeptide sequence encoded by exon 3 of a human CTLA4 gene. In anotherspecific embodiment, the CTLA4 transmembrane domain is or comprises theamino acid sequence PEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKM (inthree-letter code, Pro-Glu-Pro-Cys-Pro-Asp-Ser-Asp-Phe-Leu-Leu-Trp-Ile-Leu-Ala-Ala-Val-Ser-Ser-Gly-Leu-Phe-Phe-Tyr-Ser-Phe-Leu-Leu-Thr-Ala-Val-Ser-Leu-Ser-Lys-Met)(SEQ ID NO:1). In another specific embodiment, the CTLA4 transmembranedomain is or comprises the polypeptide sequence encoded by nucleotides610-722 of GenBank Accession No. NM_005214.4. In another specificembodiment, the CTLA4 transmembrane domain is or comprises the aminoacid sequence PDSDFLLWILAAVSSGLFFYSFLLTAVSL (in three-letter code,Pro-Asp-Ser-Asp-Phe-Leu-Leu-Trp-Ile-Leu-Ala-Ala-Val-Ser-Ser-Gly-Leu-Phe-Phe-Tyr-Ser-Phe-Leu-Leu-Thr-Ala-Val-Ser-Leu)(SEQ ID NO:2). In another specific embodiment, the CTLA4 transmembranedomain is or comprises the polypeptide sequence encoded by nucleotides636-699 of GenBank Accession No. NM_005214.4. In another specificembodiment, the CTLA4 transmembrane domain is or comprises the aminoacid sequence FLLWILAAVSSGLFFYSFLLTAV (in three-letter code,Phe-Leu-Leu-Trp-Ile-Leu-Ala-Ala-Val-Ser-Ser-Gly-Leu-Phe-Phe-Tyr-Ser-Phe-Leu-Leu-Thr-Ala-Val)(SEQ ID NO:3). See, e.g., Ensembl protein reference no.ENSP00000303939.3. In another specific embodiment, the CTLA4transmembrane domain is or comprises the polypeptide sequenceFLLWILAAVSSGLFFYSFLLT (in three-letter code,Phe-Leu-Leu-Trp-Ile-Leu-Ala-Ala-Val-Ser-Ser-Gly-Leu-Phe-Phe-Tyr-Ser-Phe-Leu-Leu-Thr)(SEQ ID NO:4), see, e.g., UNIPROT Accession No. P16410. In anotherspecific embodiment, the CTLA4 transmembrane domain is or comprises thepolypeptide sequence FLLWILVAVSLGLFFYSFLVSAVSLS (in three-letter code,Phe-Leu-Leu-Trp-Ile-Leu-Val-Ala-Val-Ser-Leu-Gly-Leu-Phe-Phe-Tyr-Ser-Phe-Leu-Val-Ser-Ala-Val-Ser-Leu-Ser)(SEQ ID NO:5). See, e.g., Shin et al., Blood 119:5678-5687 (2012). Inanother specific embodiment, the PD-1 transmembrane domain is orcomprises the amino acid sequence TLVVGVVGGLLGSLVLLVWVLAVICSRAA (inthree-letter code, Thr-Leu-Val-Val-Gly-Val-Val-Gly-Gly-Leu-Leu-Gly-Ser-Leu-Val-Leu-Leu-Val-Trp-Val-Leu-Ala-Val-Ile-Cys-Ser-Arg-Ala-Ala)(SEQ ID NO:6). See Finger et al., Gene 197(1-2):177-187 (1997). Inanother specific embodiment, the PD-1 transmembrane domain is orcomprises the amino acid sequence VGVVGGLLGSLVLLVWVLAVI (in three-lettercode,Val-Gly-Val-Val-Gly-Gly-Leu-Leu-Gly-Ser-Leu-Val-Leu-Leu-Val-Trp-Val-Leu-Ala-Val-Ile)(SEQ ID NO:7). See, e.g., UNIPROT Accession No. Q15116. In anotherspecific embodiment, the PD-1 transmembrane domain is or comprises theamino acid sequence FQTLVVGVVGGLLGSLVLLVWVLAVI (in three-letter code,Phe-Glu-Thr-Leu-Val-Val-Gly-Val-Val-Gly-Gly-Leu-Leu-Gly-Ser-Leu-Val-Leu-Leu-Val-Trp-Val-Leu-Ala-Val-Ile)(SEQ ID NO:8). See, e.g., GenBank Accession No. NM_005018.2. In certainembodiments, a nucleotide sequence that encodes one of the transmembranepolypeptides disclosed herein comprises a nucleotide sequence thatencodes any of the amino acid sequences disclosed in SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 orSEQ ID NO:8. In another specific embodiment, the PD-1 transmembranedomain is or comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 or 21 consecutive amino acids disclosed in SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQID NO:8. In certain embodiments, a nucleotide sequence that encodes oneof the polypeptides disclosed herein comprises a nucleotide sequencethat encodes at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21consecutive amino acids disclosed in SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.In constructing the polypeptide, e.g. CAR, in certain embodiments, humansequences may be combined with non-human sequences. For example, apolypeptide, e.g. CAR comprising human extracellular and intracellulardomain amino acid sequences may comprise a transmembrane domain from anon-human species; e.g., may comprise a murine CTLA4 transmembranedomain or a murine PD-1 transmembrane domain. In a more specificembodiment, the polypeptide, e.g. CAR, comprises human amino acidsequences for the extracellular and intracellular domains, and comprisesa transmembrane domain having, or consisting of, the amino acid sequenceof SEQ ID NO:5.

4.3.3. CAR Intracellular Domain

The extracellular domain of the polypeptide binds to an antigen ofinterest. In certain embodiments of any of the polypeptides describedherein, the extracellular domain comprises a receptor, or a portion of areceptor, that binds to said antigen. The extracellular domain may be,e.g., a receptor, or a portion of a receptor, that binds to saidantigen. In certain embodiments, the extracellular domain comprises, oris, an antibody or an antigen-binding portion thereof. In specificembodiments, the extracellular domain comprises, or is, a single-chainFv domain. The single-chain Fv domain can comprise, for example, a V_(L)linked to V_(H) by a flexible linker, wherein said V_(L) and V_(H) arefrom an antibody that binds said antigen.

The antigen to which the extracellular domain of the polypeptide bindscan be any antigen of interest, e.g., can be an antigen on a tumor cell.The tumor cell may be, e.g., a cell in a solid tumor, or a cell of ablood cancer. The antigen can be any antigen that is expressed on a cellof any tumor or cancer type, e.g., cells of a lymphoma, a lung cancer, abreast cancer, a prostate cancer, an adrenocortical carcinoma, a thyroidcarcinoma, a nasopharyngeal carcinoma, a melanoma, e.g., a malignantmelanoma, a skin carcinoma, a colorectal carcinoma, a desmoid tumor, adesmoplastic small round cell tumor, an endocrine tumor, an Ewingsarcoma, a peripheral primitive neuroectodermal tumor, a solid germ celltumor, a hepatoblastoma, a neuroblastoma, a non-rhabdomyosarcoma softtissue sarcoma, an osteosarcoma, a retinoblastoma, a rhabdomyosarcoma, aWilms tumor, a glioblastoma, a myxoma, a fibroma, a lipoma, or the like.In more specific embodiments, said lymphoma can be chronic lymphocyticleukemia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia,lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, splenicmarginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodalmarginal zone B cell lymphoma, MALT lymphoma, nodal marginal zone B celllymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large Bcell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascularlarge B cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma, Tlymphocyte prolymphocytic leukemia, T lymphocyte large granularlymphocytic leukemia, aggressive NK cell leukemia, adult T lymphocyteleukemia/lymphoma, extranodal NK/T lymphocyte lymphoma, nasal type,enteropathy-type T lymphocyte lymphoma, hepatosplenic T lymphocytelymphoma, blastic NK cell lymphoma, mycosis fungoides, Sezary syndrome,primary cutaneous anaplastic large cell lymphoma, lymphomatoidpapulosis, angioimmunoblastic T lymphocyte lymphoma, peripheral Tlymphocyte lymphoma (unspecified), anaplastic large cell lymphoma,Hodgkin lymphoma, or a non-Hodgkin lymphoma.

In a specific embodiment, in which the cancer is chronic lymphocyticleukemia (CLL), the B cells of the CLL have a normal karyotype. In otherspecific embodiments, in which the cancer is chronic lymphocyticleukemia (CLL), the B cells of the CLL carry a 17p deletion, an 11qdeletion, a 12q trisomy, a 13q deletion or a p53 deletion.

In certain embodiments, the antigen is a tumor-associated antigen or atumor-specific antigen. In various specific embodiments, withoutlimitation, the tumor-associated antigen or tumor-specific antigen isHer2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP),carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9,calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumorantigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD19,CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glialfibrillary acidic protein (GFAP), gross cystic disease fluid protein(GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognizedby T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA),neurofilament, neuron-specific enolase (NSE), placental alkalinephosphatase, synaptophysis, thyroglobulin, thyroid transcriptionfactor-1, the dimeric form of the pyruvate kinase isoenzyme type M2(tumor M2-PK), an abnormal ras protein, or an abnormal p53 protein.

In certain embodiments, the TAA or TSA is a cancer/testis (CT) antigen,e.g., BAGE, CAGE, CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB,MAGEC, NA88, NY-ESO-1, NY-SAR-35, OY-TES-1, SPANXB1, SPA17, SSX, SYCP1,or TPTE.

In certain other embodiments, the TAA or TSA is a carbohydrate organglioside, e.g., fuc-GM1, GM2 (oncofetal antigen-immunogenic-1;OFA-I-1); GD2 (OFA-I-2), GM3, GD3, and the like.

In certain other embodiments, the TAA or TSA is alpha-actinin-4, Bage-1,BCR-ABL, Bcr-Ab1 fusion protein, beta-catenin, CA 125, CA 15-3 (CA27.29\BCAA), CA 195, CA 242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a,CEA, coa-1, dek-can fusion protein, EBNA, EF2, Epstein Barr virusantigens, ETV6-AML1 fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205,Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, pml-RARα fusionprotein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Gage 3,4,5,6,7,GnTV, Herv-K-mel, Lage-1, NA-88, NY-Eso-1/Lage-2, SP17, SSX-2,TRP2-Int2, gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3,RAGE, GAGE-1, GAGE-2, p15(58), RAGE, SCP-1, Hom/Mel-40, PRAME, p53,H-Ras, HER-2/neu, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, humanpapillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5,MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9,CA 72-4, CAM 17.1, NuMa, K-ras, 13-Catenin, Mum-1, p16, TAGE, PSMA, CT7,telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68\KP1, CO-029,FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18,NB\70K, NY-CO-1, RCAS1, SDCCAGI6, TA-90, TAAL6, TAG72, TLP, TPS, CD19,CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growthfactor variant III), sperm protein 17 (Sp17), mesothelin, PAP (prostaticacid phosphatase), prostein, TARP (T cell receptor gamma alternatereading frame protein), Trp-p8, STEAP1 (six-transmembrane epithelialantigen of the prostate 1), an abnormal ras protein, or an abnormal p53protein. In another specific embodiment, said tumor-associated antigenor tumor-specific antigen is integrin ανβ3 (CD61), galactin, K-Ras(V-Ki-ras2 Kirsten rat sarcoma viral oncogene), or Ral-B. Othertumor-associated and tumor-specific antigens are known to those in theart.

Antibodies, and scFvs, that bind to TSAs and TAAs are known in the art,as are nucleotide sequences that encode them.

In certain specific embodiments, the antigen is an antigen notconsidered to be a TSA or a TAA, but which is nevertheless associatedwith tumor cells, or damage caused by a tumor. In certain embodiments,for example, the antigen is, e.g., a growth factor, cytokine orinterleukin, e.g., a growth factor, cytokine, or interleukin associatedwith angiogenesis or vasculogenesis. Such growth factors, cytokines, orinterleukins can include, e.g., vascular endothelial growth factor(VEGF), basic fibroblast growth factor (bFGF), platelet-derived growthfactor (PDGF), hepatocyte growth factor (HGF), insulin-like growthfactor (IGF), or interleukin-8 (IL-8). Tumors can also create a hypoxicenvironment local to the tumor. As such, in other specific embodiments,the antigen is a hypoxia-associated factor, e.g., HIF-1α, HIF-1β,HIF-2α, HIF-2β, HIF-3α, or HIF-3β. Tumors can also cause localizeddamage to normal tissue, causing the release of molecules known asdamage associated molecular pattern molecules (DAMPs; also known asalarmins). In certain other specific embodiments, therefore, the antigenis a DAMP, e.g., a heat shock protein, chromatin-associated protein highmobility group box 1 (HMGB1), S100A8 (MRP8, calgranulin A), S100A9(MRP14, calgranulin B), serum amyloid A (SAA), or can be adeoxyribonucleic acid, adenosine triphosphate, uric acid, or heparinsulfate.

In certain embodiments of the polypeptides described herein, theextracellular domain is joined to said transmembrane domain by a linker,spacer or hinge polypeptide sequence, e.g., a sequence from CD28 or asequence from CTLA4.

4.3.4. Bispecific CARs

In certain embodiments of the T lymphocytes or methods described herein,the T lymphocytes, in addition to comprising a cell death polypeptide,comprise two or more CARs in which the primary signaling mechanism andcostimulatory mechanism are split into two or more polypeptides.

In certain embodiments, for example, the T lymphocytes comprise a celldeath polypeptide, and at least two different other polypeptides, e.g.,chimeric receptors, in which the immune signal derived from binding of aprimary signaling polypeptide, e.g., chimeric receptor, to a firstantigen is separated from a costimulatory signal produced by acostimulatory polypeptide, e.g., chimeric receptor, wherein thecostimulatory signal is dependent on antigen binding of a second antigenby the second chimeric receptor.

In one embodiment, the T lymphocyte comprises a primary signalingpolypeptide comprising a first extracellular antigen binding domain thatbinds a first antigen, and a first intracellular signaling domain,wherein said primary signaling polypeptide does not comprise aco-stimulatory domain; and a co-stimulatory comprising a secondextracellular antigen binding domain binding a second antigen, or areceptor that binds said second antigen; and a second intracellularsignaling domain; wherein said T lymphocyte becomes maximally cytotoxiconly when said first signaling domain and said second signaling domainare both activated by said first antigen and said second antigen,respectively. In a specific embodiment, binding of said first antigen tosaid first antigen binding domain without binding of said second antigento said second binding domain, or binding of said second antigen to saidsecond antigen binding domain without binding of first second antigen tosaid first binding domain, induces anergy of said T lymphocyte, ornon-responsiveness of said T-lymphocyte to said first antigen or saidsecond antigen.

In another specific embodiment, said first antigen binding domain andsaid second antigen binding domain are independently an antigen-bindingportion of a receptor, an antigen-binding portion of an antibody, orother peptide-based macromolecular antigen binding agent. In certainspecific embodiments, either or both of said first antigen bindingdomain or said second antigen binding domain are scFv antibodyfragments. In specific embodiments, either or both of said primarysignaling polypeptide or said co-stimulatory polypeptide additionallycomprise a transmembrane domain. In other specific embodiments, saidprimary signaling polypeptide or said co-stimulatory polypeptidecomprises a T lymphocyte survival motif. In a specific embodiment, the Tlymphocyte survival motif is a CD28 T lymphocyte survival motif. Inother specific embodiments, said T lymphocyte survival motif is anintracellular signaling domain of IL-7 receptor (IL-7R), anintracellular signaling domain of IL-12 receptor, an intracellularsignaling domain of IL-15 receptor, an intracellular signaling domain ofIL-21 receptor, or an intracellular signaling domain of transforminggrowth factor β (TGFβ) receptor. In another more specific embodiment,said primary signaling polypeptide or said co-stimulatory polypeptidecomprise a portion of a CD28 molecule that comprises a T lymphocytesurvival motif. In a more specific embodiment, said primary signalingpolypeptide or said co-stimulatory polypeptide comprise a CD28 moleculethat comprises a T lymphocyte survival motif In certain specificembodiments, said first intracellular signaling domain comprises apolypeptide sequence comprising an immunoreceptor tyrosine-basedactivation motif (ITAM). In a more specific embodiment, said polypeptidesequence is a CD3ζ signaling domain.

In certain specific embodiments, said first antigen is an antigen on atumor cell. In a more specific embodiment, said tumor cell is a cell ina solid tumor. In another more specific embodiment, said tumor cell is ablood cancer cell. In another specific embodiment, said antigen is atumor-associated antigen or a tumor-specific antigen. In more specificembodiments, said tumor-associated antigen or tumor-specific antigen isHer2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP),carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9,calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumorantigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34,CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillaryacidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15),HMB-45 antigen, protein melan-A (melanoma antigen recognized by Tlymphocytes; MART-1), myo-D1, muscle-specific actin (MSA),neurofilament, neuron-specific enolase (NSE), placental alkalinephosphatase, synaptophysin, thyroglobulin, thyroid transcriptionfactor-1, the dimeric form of the pyruvate kinase isoenzyme type M2(tumor M2-PK), an abnormal ras protein, or an abnormal p53 protein.

In another specific embodiment, said second antigen is a growth factor,cytokine, or interleukin. The second antigen is a growth factor,cytokine, or interleukin associated with angiogenesis or vasculogenesis.In more specific embodiments, said second antigen is vascularendothelial growth factor (VEGF), basic fibroblast growth factor (bFGF),platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF),insulin-like growth factor (IGF), or interleukin-8 (IL-8).

In another specific embodiment, signal transduction activation providedby said second antigen is non-antigenic, but is associated with hypoxia.In more specific embodiments, said stimulus is induced by activation ofhypoxia-inducible factor-1α (HIF-1α), HIF-1β, HIF-2α, HIF-2β, HIF-3α, orHIF-3β.

In another specific embodiment, said second antigen is an interleukin.

In another specific embodiment, said second antigen is a damageassociated molecular pattern molecule (DAMP; also known as an alarmin).In more specific embodiments, said DAMP is a heat shock protein,chromatin-associated protein high mobility group box 1 (HMGB1), S100A8(also known as MRP8, or calgranulin A), S100A9 (also known as MRP14, orcalgranulin B), serum amyloid A (SAA), deoxyribonucleic acid, adenosinetriphosphate, uric acid, or heparin sulfate.

In certain specific embodiments, said second antigen is an antigen on anantibody that binds to an antigen presented by a tumor cell.

In a specific embodiment of any of the embodiments herein, saidco-stimulatory polypeptide comprises one or more co-stimulatory domains.In specific embodiments, said one or more co-stimulatory domainscomprises one or more of a co-stimulatory CD27 polypeptide sequence, aco-stimulatory CD28 polypeptide sequence, a co-stimulatory OX40 (CD134)polypeptide sequence, a co-stimulatory 4-1BB (CD137) polypeptidesequence, or a co-stimulatory inducible T-cell co-stimulatory (ICOS)polypeptide sequence.

In a specific embodiment, said primary signaling polypeptide comprisesan extracellular tumor antigen-binding domain and a CD3ζ signalingdomain, and wherein said co-stimulatory polypeptide comprises anantigen-binding domain wherein said antigen is an angiogenic orvasculogenic factor, and one or more co-stimulatory molecule signalingdomains. Said angiogenic factor can be, e.g., VEGF. Said one or moreco-stimulatory molecule signaling motifs can comprise, e.g.,co-stimulatory signaling domains from each of CD28, OX40, and 4-1BB. Ina more specific embodiment, said primary signaling polypeptide comprisesan extracellular tumor antigen-binding domain and a CD3ζ signalingdomain, and wherein said co-stimulatory polypeptide comprises anantigen-binding domain wherein said antigen is VEGF, and co-stimulatorysignaling domains from each of CD28, OX40, and 4-1BB.

In a more specific embodiment, said primary signaling polypeptide orsaid co-stimulatory polypeptide comprises a T lymphocyte survival motif.In more specific embodiments, said T lymphocyte survival motif is, or isderived from, an intracellular signaling domain of IL-7 receptor(IL-7R), an intracellular signaling domain of IL-12 receptor, anintracellular signaling domain of IL-15 receptor, an intracellularsignaling domain of IL-21 receptor, or an intracellular signaling domainof transforming growth factor β (TGFβ) receptor. In a more specificembodiment of said T lymphocyte, therefore, said primary signalingpolypeptide comprises an extracellular tumor antigen-binding domain anda CD3ζ signaling domain, and wherein said co-stimulatory polypeptidecomprises an antigen-binding domain wherein said antigen is VEGF, anIL-7 receptor intracellular T lymphocyte survival motif, andco-stimulatory signaling domains from each of CD28, OX40, and 4-1BB.

In another specific embodiment of the T lymphocyte, said first antigenis a tumor-specific antigen or a tumor-associated antigen, and saidfirst intracellular signaling domain comprises a CD3ζ signaling domain;and wherein said co-stimulatory polypeptide comprises an antigen-bindingdomain that binds said second antigen, and co-stimulatory signalingdomains from each of CD28, OX40, and 4-1BB. In a more specificembodiment, said co-stimulatory polypeptide further comprises anintracellular T lymphocyte survival motif, e.g., a T lymphocyte survivalmotif that is, or is derived from, an intracellular signaling domain ofIL-7 receptor (IL-7R), an intracellular signaling domain of IL-12receptor, an intracellular signaling domain of IL-15 receptor, anintracellular signaling domain of IL-21 receptor, or an intracellularsignaling domain of transforming growth factor β (TGFβ) receptor.

In a specific embodiment of any of the T lymphocytes provided herein,said second antigen is VEGF or IL-4.

In another aspect, provided herein is a T lymphocyte comprising a celldeath polypeptide, a co-stimulatory polypeptide comprising a firstextracellular antigen binding domain that binds a first antigen, and afirst intracellular signaling domain; and a primary signalingpolypeptide comprising a second extracellular antigen binding domainbinding a second antigen, or a receptor that binds said second antigen;and a second intracellular signaling domain, wherein said primarysignaling polypeptide does not comprise a co-stimulatory domain; whereinsaid modified lymphocyte becomes maximally cytotoxic only when saidfirst signaling domain and said second signaling domain are bothactivated by said first antigen and said second antigen, respectively.In a specific embodiment, binding of said first antigen to said firstantigen binding domain without binding of said second antigen to saidsecond binding domain, or binding of said second antigen to said secondantigen binding domain without binding of first second antigen to saidfirst binding domain induces anergy of said T lymphocyte, ornon-responsiveness of said T lymphocyte to said first antigen. In aspecific embodiment, said first antigen-binding domain and saidantigen-binding domain are independently an antigen-binding portion of areceptor or an antigen-binding portion of an antibody. In anotherspecific embodiment, either or both of said first antigen binding domainor said second antigen binding domain are scFv antibody fragments. Inspecific embodiments, said co-stimulatory polypeptide and/or saidprimary signaling polypeptide additionally comprise a transmembranedomain. In a more specific embodiment, said co-stimulatory polypeptideor said primary signaling polypeptide comprises a T lymphocyte survivalmotif, e.g., any of the T lymphocyte survival motifs described herein.In another specific embodiment, said first antigen is an antigen on atumor cell, e.g., a cell in a solid tumor or a blood cancer cell. In aspecific embodiment, said first antigen is a tumor-associated antigen ora tumor-specific antigen, e.g., Her2, prostate stem cell antigen (PSCA),alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancerantigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membraneprotein (EMA), epithelial tumor antigen (ETA), tyrosinase,melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117,chromogranin, cytokeratin, desmin, glial fibrillary acidic protein(GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen,protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1),myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specificenolase (NSE), placental alkaline phosphatase, synaptophysin,thyroglobulin, thyroid transcription factor-1, the dimeric form of thepyruvate kinase isoenzyme type M2 (tumor M2-PK), an abnormal rasprotein, an abnormal p53 protein, CD19, CD22, CD27, CD30, CD70, GD2(ganglioside G2), EGFRvIII (epidermal growth factor variant III), spermprotein 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase),prostein, TARP (T cell receptor gamma alternate reading frame protein),Trp-p8, or STEAP1 (six-transmembrane epithelial antigen of the prostate1). In another specific embodiment, said tumor-associated antigen ortumor-specific antigen is integrin ανβ3 (CD61), galactin, K-Ras(V-Ki-ras2 Kirsten rat sarcoma viral oncogene), or Ral-B.

In certain specific embodiments, said second intracellular signalingdomain comprises a polypeptide sequence comprising an immunoreceptortyrosine-based activation motif (ITAM), e.g., a CD3ζ signaling domain.In a specific embodiment, said second antigen is a growth factor,cytokine, or interleukin. In another specific embodiment, said secondantigen is a growth factor, cytokine, or interleukin associated withangiogenesis or vasculogenesis, e.g., VEGF, bFGF, PDGF, HGF, IGF, orIL-8. In other more specific embodiments, signal transduction by saidsecond chimeric receptor is induced by activation of ahypoxia-associated factor, e.g., HIF-1α, HIF-1β, HIF-2α, HIF-2β, HIF-3α,or HIF-3β. In other specific embodiments, said second antigen is aninterleukin. In other specific embodiments, said second antigen is aDAMP, e.g., a heat shock protein, HMGB1, S100A8, S100A9, SAA, DNA, ATP,uric acid, or heparin sulfate. In other specific embodiments, saidsecond antigen is an administered peptide, e.g., an antibody or asynthetic polypeptide. In other specific embodiments, said secondantigen is an antigen on an antibody that binds to an antigen presentedby a tumor cell. In certain specific embodiments, said co-stimulatorypolypeptide comprises one or more co-stimulatory domains, e.g., one ormore of a co-stimulatory CD27 polypeptide sequence, a co-stimulatoryCD28 polypeptide sequence, a co-stimulatory OX40 (CD134) polypeptidesequence, a co-stimulatory 4-1BB (CD137) polypeptide sequence, or aco-stimulatory inducible T-cell co-stimulatory (ICOS) polypeptidesequence. In any of the above embodiments, in a specific embodiment,said co-stimulatory polypeptide or said primary signaling polypeptidecomprises a T lymphocyte survival motif, e.g., said T lymphocytesurvival motif is, or is derived from, an intracellular signaling domainof IL-7 receptor (IL-7R), an intracellular signaling domain of IL-12receptor, an intracellular signaling domain of IL-15 receptor, anintracellular signaling domain of IL-21 receptor, or an intracellularsignaling domain of transforming growth factor β (TGFβ) receptor.

4.4. Isolated Polypeptides

Any of the polypeptides, comprising a CTLA4 or PD-1 transmembranedomain, provided herein, may be modified by, e.g., acylation, amidation,glycosylation, methylation, phosphorylation, sulfation, sumoylation,ubiquitylation, or the like. The polypeptides may be labeled with alabel capable of providing a detectable signal, e.g., with radioisotopesand fluorescent compounds. One or more side chains of the first orsecond polypeptides may be derivatized, e.g., derivatization of lysinyland amino terminal residues with succinic or other carboxylic acidanhydrides, or derivatization with, e.g., imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; andtransaminase-catalyzed reaction with glyoxylate. Carboxyl side groups,aspartyl or glutamyl, may be selectively modified by reaction withcarbodiimides (R—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide.

4.5. Isolated Nucleic Acids

The polypeptides provided herein (e.g., chimeric receptors) can beencoded by polynucleotide sequences according to well-known methods inthe art. The polynucleotides may be contained within any polynucleotidevector suitable for the transformation of immune cells, e.g., Tlymphocytes. For example, T lymphocytes may be transformed usingsynthetic vectors, lentiviral or retroviral vectors, autonomouslyreplicating plasmids, a virus (e.g., a retrovirus, lentivirus,adenovirus, or herpes virus), or the like, containing polynucleotidesencoding the first and second polypeptides (e.g., chimeric receptors).Lentiviral vectors suitable for transformation of T lymphocytes include,but are not limited to, e.g., the lentiviral vectors described in U.S.Pat. Nos. 5,994,136; 6,165,782; 6,428,953; 7,083,981; and 7,250,299, thedisclosures of which are hereby incorporated by reference in theirentireties. HIV vectors suitable for transformation of T lymphocytesinclude, but are not limited to, e.g., the vectors described in U.S.Pat. No. 5,665,577, the disclosure of which is hereby incorporated byreference in its entirety.

Nucleic acids useful in the production of the first and secondpolypeptides, e.g., within a T lymphocyte, include DNA, RNA, or nucleicacid analogs. Nucleic acid analogs can be modified at the base moiety,sugar moiety, or phosphate backbone, and can include deoxyuridinesubstitution for deoxythymidine, 5-methyl-2′-deoxycytidine or5-bromo-2′-deoxycytidine substitution for deoxycytidine. Modificationsof the sugar moiety can include modification of the 2′ hydroxyl of theribose sugar to form 2′-O-methyl or 2′-O-allyl sugars. The deoxyribosephosphate backbone can be modified to produce morpholino nucleic acids,in which each base moiety is linked to a six membered, morpholino ring,or peptide nucleic acids, in which the deoxyphosphate backbone isreplaced by a pseudopeptide backbone and the four bases are retained.See, for example, Summerton and Weller (1997) Antisense Nucleic AcidDrug Dev. 7:187-195; and Hyrup et al. (1996) Bioorgan. Med. Chain.4:5-23. In addition, the deoxyphosphate backbone can be replaced with,for example, a phosphorothioate or phosphorodithioate backbone, aphosphoroamidite, or an alkyl phosphotriester backbone.

4.6. Cells

Non-limiting examples of cells in which the cell death polypeptides andmultimerizing or dimerizing agents may be used include, but are notlimited to, natural killer (NK) cells, dendritic cells (DC), placentalstem cells (e.g., the placental stem cells disclosed in U.S. Pat. Nos.7,468,276; 8,057,788 and 8,202,703, the disclosures of which are herebyincorporated by reference in their entireties), mesenchymal-like stemcells from umbilical cord blood, placental blood, peripheral blood, bonemarrow, dental pulp, adipose tissue, osteochondral tissue, and the like;embryonic stem cells, embryonic germ cells, neural crest stem cells,neural stem cells, and differentiated cells (e.g., fibroblasts, etc.).The cell death polypeptides, and multimerizing or dimerizing agents, mayalso be used in tumor cell lines, e.g., for animal model experimentalpurposes.

In a specific embodiment, the cells comprising the polypeptides providedherein are T lymphocytes. The T lymphocytes comprising the polypeptidesprovided herein may be naive T lymphocytes or MHC-restricted Tlymphocytes. In certain embodiments, the T lymphocytes are tumorinfiltrating lymphocytes (TILs). In certain embodiments, the Tlymphocytes have been isolated from a tumor biopsy, or have beenexpanded from T lymphocytes isolated from a tumor biopsy. In certainother embodiments, the T lymphocytes have been isolated from, or areexpanded from T lymphocytes expanded from, peripheral blood, cord blood,or lymph.

The immune cells, e.g., T lymphocytes, used in the present methods arepreferably autologous to an individual to whom the T lymphocytes are tobe administered. In certain other embodiments, the T lymphocytes areallogeneic to an individual to whom the T lymphocytes are to beadministered. Where allogeneic T lymphocytes are used to prepare Tlymphocytes, it is preferable to select T lymphocytes that will reducethe possibility of graft-versus-host disease (GVHD) in the individual.For example, in certain embodiments, virus-specific T lymphocytes areselected for preparation of T lymphocytes; such lymphocytes will beexpected to have a greatly reduced native capacity to bind to, and thusbecome activated by, any recipient antigens. In certain embodiments,recipient-mediated rejection of allogeneic T lymphocytes can be reducedby co-administration to the host of one or more immunosuppressiveagents, e.g., cyclosporine, tacrolimus, sirolimus, cyclophosphamide, orthe like.

In one embodiment, T lymphocytes are obtained from an individual,optionally then expanded, and then transformed with a polynucleotideencoding a cell death polypeptide, and optionally one or morepolynucleotides encoding one or more CAR(s), and optionally thenexpanded. In another embodiment, T lymphocytes are obtained from anindividual, optionally then expanded, and then transformed with apolynucleotide encoding a cell death polypeptide, and optionally one ormore polynucleotides encoding one or more CAR(s), and optionally thenexpanding. Cells containing any of the polynucleotide may be selectedusing one or more selectable markers.

In certain embodiments, any of the T lymphocytes provided herein expressor comprise native TCR proteins, e.g., TCR-α and TCR-β that are capableof forming native TCR complexes, in addition to the CTLA4 or PD-1transmembrane domain-containing polypeptide. In certain otherembodiments, either or both of the native genes encoding TCR-α and TCR-βin the T lymphocytes are modified to be non-functional, e.g., a portionor all are deleted, a mutation is inserted, etc.

In certain embodiments, any of the T lymphocytes provided herein areisolated from a tumor lesion, e.g., are tumor-infiltrating lymphocytes;such T lymphocytes are expected to be specific for a TSA or TAA.

T lymphocytes, and T lymphocytes comprising a polypeptide comprising aCD3ζ signaling domain and a CD28 co-stimulatory domain can be expandedusing antibodies to CD3 and CD28, e.g., antibodies attached to beads, orto the surface of a cell culture plate; see, e.g., U.S. Pat. Nos.5,948,893; 6,534,055; 6,352,694; 6,692,964; 6,887,466; and 6,905,681.

In any of the above embodiments, the antigen and/or antibody can existfree in the medium in which the T lymphocytes are cultures, or either orboth can be attached to a solid support, e.g., tissue culture plasticsurface, beads, or the like.

The T lymphocytes provided herein can optionally comprise a second typeof “suicide gene” or “safety switch”, in addition to the cell deathpolypeptide. For example, the T lymphocytes, in certain embodiments, cancomprise an HSV thymidine kinase gene (HSV-TK), which causes death ofthe T lymphocytes upon contact with gancyclovir. In another embodiment,the T lymphocytes express or comprise an inducible caspase, e.g., aninducible caspase 9 (icaspase9), e.g., a fusion protein between caspase9 and human FK506 binding protein allowing for dimerization using aspecific small molecule pharmaceutical. See Straathof et al., Blood105(11):4247-4254 (2005).

4.7. Methods of Using Cells Comprising Cell Death Polypeptides

The cells, e.g., T lymphocytes, provided herein that comprise cell deathpolypeptides and optionally one or more CARs, as described elsewhereherein, can be used to treat an individual having one or more types ofcells desired to be targeted by the cells described herein, e.g., one ormore types of cells to be killed. In certain embodiments, the cells tobe killed are cancer cells, e.g., tumor cells. In specific embodiments,the cancer cells are cells of a solid tumor. In specific embodiments,the cells are cells of a lymphoma, a lung cancer, a breast cancer, aprostate cancer, an adrenocortical carcinoma, a thyroid carcinoma, anasopharyngeal carcinoma, a melanoma, e.g., a malignant melanoma, a skincarcinoma, a colorectal carcinoma, a desmoid tumor, a desmoplastic smallround cell tumor, an endocrine tumor, an Ewing sarcoma, a peripheralprimitive neuroectodermal tumor, a solid germ cell tumor, ahepatoblastoma, a neuroblastoma, a non-rhabdomyosarcoma soft tissuesarcoma, an osteosarcoma, a retinoblastoma, a rhabdomyosarcoma, a Wilmstumor, a glioblastoma, a myxoma, a fibroma, a lipoma, or the like. Inmore specific embodiments, said lymphoma can be chronic lymphocyticleukemia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia,lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, splenicmarginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodalmarginal zone B cell lymphoma, MALT lymphoma, nodal marginal zone B celllymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large Bcell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascularlarge B cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma, Tlymphocyte prolymphocytic leukemia, T lymphocyte large granularlymphocytic leukemia, aggressive NK cell leukemia, adult T lymphocyteleukemia/lymphoma, extranodal NK/T lymphocyte lymphoma, nasal type,enteropathy-type T lymphocyte lymphoma, hepatosplenic T lymphocytelymphoma, blastic NK cell lymphoma, mycosis fungoides, Sezary syndrome,primary cutaneous anaplastic large cell lymphoma, lymphomatoidpapulosis, angioimmunoblastic T lymphocyte lymphoma, peripheral Tlymphocyte lymphoma (unspecified), anaplastic large cell lymphoma,Hodgkin lymphoma, or a non-Hodgkin lymphoma.

In certain embodiments, when the modified cells, e.g., modified Tlymphocytes described herein are administered to a subject in needthereof, the combination of multimerizing agent and cell deathpolypeptide selected are chosen such that they are compatible with thepatient population (or subpopulation) in which the cells, e.g., Tlymphocytes, have been administered. By way of example only, if themultimerizing agent selected is the antibody rituximab, then in certainembodiments the patient population is individuals having a cancer of theB cells, e.g., B cell lymphoma.

Efficacy of the cells, e.g., T lymphocytes, after administration to anindividual having a disease or disorder remediable by such cells, e.g.,T lymphocytes, e.g., an individual having cancer, can be assessed by oneor more criteria, specific to the particular disease or disorder, knownto those of ordinary skill in the art, to be indicative of progress ofthe disease or disorder. Generally, administration of the cells to suchan individual is effective when one or more of said criteria detectably,e.g., significantly, moves from a disease state value or range to, ortowards, a normal value or range.

The cells, e.g., T lymphocytes, may be formulated in anypharmaceutically-acceptable solution, preferably a solution suitable forthe delivery of living cells, e.g., saline solution (such as Ringer'ssolution), gelatins, carbohydrates (e.g., lactose, amylose, starch, orthe like), fatty acid esters, hydroxymethylcellulose, polyvinylpyrolidine, etc. Such preparations are preferably sterilized prior toaddition of the cells, and may be mixed with auxiliary agents such aslubricants, preservatives, stabilizers, emulsifiers, salts forinfluencing osmotic pressure, buffers, and coloring. Pharmaceuticalcarriers suitable for use in formulating the cells are known in the artand are described, for example, in WO 96/05309.

In certain embodiments, the cells, e.g., T lymphocytes, are formulatedinto individual doses, wherein said individual doses comprise at least,at most, or about 1×10⁴, 5×10⁴, 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷,5×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰ or 1×10¹¹ Tlymphocytes. In certain embodiments, the cells are formulated forintravenous, intraarterial, parenteral, intramuscular, subcutaneous,intrathecal, or intraocular administration, or administration within aparticular organ or tissue.

5. EXAMPLES 5.1. Example 1: Treatment of B Cell Lymphoma

An individual presents with B-cell chronic lymphocytic leukemia, a Bcell lymphoma. Testing of B cells from the individual determines thatthe B cells carry a 17p deletion. T lymphocytes are obtained from theindividual, transfected with a lentiviral vector comprising a nucleotidesequence that encodes a chimeric antigen receptor (CAR), and transfectedwith a second lentiviral vector comprising a nucleotide sequenceencoding a dimerizable cell death polypeptide comprising anextracellular domain that comprises a mimotope that can be bound by theantibody rituximab, and an intracellular domain that comprises a caspase9 domain. The T lymphocytes are expanded using CD3+CD28-coated beads tosufficient numbers for administration. The chimeric receptor comprisesan extracellular antigen-binding region that binds to CD19; atransmembrane domain from CTLA4; intracellular co-stimulatory domainfrom CD28; and an intracellular CD3ζ domain. The individual isadministered between 10⁹ and 10¹⁰ of the T lymphocytes in 200 mL salinesolution by intravenous infusion over 30 minutes. The individual ismonitored for two weeks afterwards to establish a reduction of at least90% of CD19+ B cells in the individual's blood. Where the patient, afteradministration of the T lymphocytes, shows signs of distress due to theT lymphocytes (e.g., difficulty breathing, fever, abnormal serumcytokine levels, rash, or the like), rituximab is administered at adosage of 200-500 mg/m² or until symptoms abate.

5.2. Example 2: Treatment of a B Cell Lymphoma

An individual presents with B-cell chronic lymphocytic leukemia, a Bcell lymphoma. Testing of B cells from the individual determines thatthe B cells carry a 17p deletion. About 10⁶ T lymphocytes are obtainedfrom the individual, transfected with a lentiviral vector comprising anucleotide sequence encoding a cell death polypeptide comprising anextracellular domain that comprises a mimotope that can be bound by theantibody rituximab and an intracellular domain that comprises a caspase8 domain, and transfected with a lentiviral vector comprising anucleotide sequence that encodes a CAR. The CAR comprises anextracellular antigen-binding region that binds to CD19; a transmembranedomain from PD-1; intracellular co-stimulatory domain from CD28; and anintracellular CD3 domain. CAR-expressing T lymphocytes are administeredto the individual without prior expansion of the T lymphocytes. Theindividual is administered between 10⁵ and 10⁶ of the T lymphocytes in200 mL saline solution by intravenous infusion over 30 minutes. Theindividual is monitored for two weeks afterwards to establish areduction of at least 90% of CD19+ B cells in the individual's blood.Where the patient, after administration of the T lymphocytes, showssigns of distress due to the T lymphocytes (e.g., difficulty breathing,fever, abnormal serum cytokine levels, rash, or the like), rituximab isadministered at a dosage of 200-500 mg/m² or until symptoms abate.

5.3. Example 3: Treatment of B Cell Lymphoma

An individual presents with B-cell chronic lymphocytic leukemia, a Bcell lymphoma. Testing of B cells from the individual determines thatthe B cells carry a p53 deletion. T lymphocytes are obtained from theindividual, transfected with a lentiviral vector comprising a nucleotidesequence encoding a cell death polypeptide comprising an extracellulardomain that comprises an epitope that can be bound by the antibodyrituximab and an intracellular domain that comprises a caspase 3 domain,and transfected with a lentiviral vector comprising a nucleotidesequence that encodes a CAR. The T lymphocytes are expanded usingCD3+CD28-coated beads to sufficient numbers for administration. The CARcomprises an extracellular antigen-binding region that binds to CD19; atransmembrane domain from CTLA4; intracellular co-stimulatory domainsfrom each of CD28, 4-1BB, and OX40; and an intracellular CD3ζ domain.The individual is administered between 10⁹ and 10¹⁰ of the T lymphocytesin 200 mL saline solution by intravenous infusion over 30 minutes. Theindividual is monitored for two weeks afterwards to establish areduction of at least 90% of CD19+ B cells in the individual's blood.Where the patient, after administration of the T lymphocytes, showssigns of distress due to the T lymphocytes (e.g., difficulty breathing,fever, abnormal serum cytokine levels, rash, or the like), rituximab isadministered at a dosage of 200-500 mg/m² or until symptoms abate.

5.4. Example 4: Treatment of a B Cell Lymphoma

An individual presents with B-cell chronic lymphocytic leukemia, a Bcell lymphoma. Testing of B cells from the individual determines thatthe B cells carry a p53 deletion. About 10⁶ T lymphocytes are obtainedfrom the individual, transfected with a lentiviral vector comprising anucleotide sequence encoding a cell death polypeptide comprising anextracellular domain that comprises an epitope that can be bound by theantibody rituximab and an intracellular domain that comprises a caspase9 domain, and transfected with a lentiviral vector comprising anucleotide sequence that encodes a CAR. The CAR comprises anextracellular antigen-binding region that binds to CD19; a transmembranedomain from PD-1; intracellular co-stimulatory domains from each ofCD28, 4-1BB, and OX40; and an intracellular CD3ζ domain. CAR-expressingT lymphocytes are administered to the individual without prior expansionof the T lymphocytes. The individual is administered between 10⁵ and 10⁶of the T lymphocytes in 200 mL saline solution by intravenous infusionover 30 minutes. The individual is monitored for two weeks afterwards toestablish a reduction of at least 90% of CD19+ B cells in theindividual's blood. Where the patient, after administration of the Tlymphocytes, shows signs of distress due to the T lymphocytes (e.g.,difficulty breathing, fever, abnormal serum cytokine levels, rash, orthe like), rituximab is administered at a dosage of 200-500 mg/m² oruntil symptoms abate.

5.5. Example 5: Treatment of Prostate Cancer

An individual presents with stage T2 prostate cancer, with no spread toregional or other lymph nodes (N0, M0). Histological grade is determinedto be G2. Overall, the individual is determined to have Stage IIprostate cancer. The individual is administered between 10⁹ and 10¹⁰ Tlymphocytes that comprise a CAR, in 200 mL saline solution byintravenous infusion over 30 minutes. The CAR comprises an extracellularantigen-binding region that binds to PSCA, a transmembrane domain fromCTLA4, intracellular co-stimulatory domain from CD28, and anintracellular CD3ζ domain. The T lymphocytes also comprise a cell deathpolypeptide comprising an extracellular domain that comprises an epitopethat can be bound by the antibody rituximab, and an intracellular domainthat comprises a caspase 3, caspase 8, or caspase 9 domain. Theindividual is re-assessed for prostate cancer stage and spread to lymphnodes, and histology of biopsied prostate tissue is performed, at 30, 60and 90 days post-administration. Where the patient, after administrationof the T lymphocytes, shows signs of distress due to the T lymphocytes(e.g., difficulty breathing, fever, abnormal serum cytokine levels,rash, or the like), rituximab is administered at a dosage of 200-500mg/m² or until symptoms abate.

5.6. Example 6: Treatment of Prostate Cancer

An individual presents with stage T2 prostate cancer, with no spread toregional or other lymph nodes (N0, M0). Histological grade is determinedto be G2. Overall, the individual is determined to have Stage IIprostate cancer. The individual is administered between 10⁹ and 10¹⁰ Tlymphocytes that comprise a CAR, in 200 mL saline solution byintravenous infusion over 30 minutes. The CAR comprises an extracellularantigen-binding region that binds to PSCA, a transmembrane domain fromPD-1, intracellular co-stimulatory domain from CD28, and anintracellular CD3ζ domain. The T lymphocytes also comprise a cell deathpolypeptide comprising an extracellular domain that comprises an epitopethat can be bound by the antibody rituximab, and an intracellular domainthat comprises a caspase 3, caspase 8, or caspase 9 domain. Theindividual is re-assessed for prostate cancer stage and spread to lymphnodes, and histology of biopsied prostate tissue is performed, at 30, 60and 90 days post-administration. Where the patient, after administrationof the T lymphocytes, shows signs of distress due to the T lymphocytes(e.g., difficulty breathing, fever, abnormal serum cytokine levels,rash, or the like), rituximab is administered at a dosage of 200-500mg/m² or until symptoms abate.

5.7. Example 7: Treatment of Prostate Cancer

An individual presents with stage T2 prostate cancer, with no spread toregional or other lymph nodes (N0, M0). Histological grade is determinedto be G2. Overall, the individual is determined to have Stage IIprostate cancer. The individual is administered between 10⁹ and 10¹⁰ Tlymphocytes that comprise a CAR, in 200 mL saline solution byintravenous infusion over 30 minutes. The CAR comprises an extracellularantigen-binding region that binds to PSCA, a transmembrane domain fromCTLA-4, intracellular co-stimulatory domains from each of CD28, 4-1BB,and OX40, and an intracellular CD3ζ domain. The T lymphocytes alsocomprise a cell death polypeptide comprising an extracellular domainthat comprises an epitope that can be bound by the antibody rituximab,and an intracellular domain that comprises a caspase 3, caspase 8, orcaspase 9 domain. The individual is re-assessed for prostate cancerstage and spread to lymph nodes, and histology of biopsied prostatetissue is performed, at 30, 60 and 90 days post-administration. Wherethe patient, after administration of the T lymphocytes, shows signs ofdistress due to the T lymphocytes (e.g., difficulty breathing, fever,abnormal serum cytokine levels, rash, or the like), rituximab isadministered at a dosage of 200-500 mg/m² or until symptoms abate.

5.8. Example 8: Treatment of Prostate Cancer

An individual presents with stage T2 prostate cancer, with no spread toregional or other lymph nodes (N0, M0). Histological grade is determinedto be G2. Overall, the individual is determined to have Stage IIprostate cancer. The individual is administered between 10⁹ and 10¹⁰ Tlymphocytes that comprise a CAR, in 200 mL saline solution byintravenous infusion over 30 minutes. The CAR comprises an extracellularantigen-binding region that binds to PSCA, a transmembrane domain fromPD-1, intracellular co-stimulatory domains from each of CD28, 4-1BB, andOX40, and an intracellular CD3ζ domain. The T lymphocytes also comprisea cell death polypeptide comprising an extracellular domain thatcomprises an epitope that can be bound by the antibody rituximab, and anintracellular domain that comprises a caspase 3, caspase 8, or caspase 9domain. The individual is re-assessed for prostate cancer stage andspread to lymph nodes, and histology of biopsied prostate tissue isperformed, at 30, 60 and 90 days post-administration. Where the patient,after administration of the T lymphocytes, shows signs of distress dueto the T lymphocytes (e.g., difficulty breathing, fever, abnormal serumcytokine levels, rash, or the like), rituximab is administered at adosage of 200-500 mg/m² or until symptoms abate.

EQUIVALENTS

The present disclosure is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of thesubject matter provided herein, in addition to those described, willbecome apparent to those skilled in the art from the foregoingdescription. Such modifications are intended to fall within the scope ofthe appended claims.

Various publications, patents and patent applications are cited herein,the disclosures of which are incorporated by reference in theirentireties.

What is claimed is:
 1. A T lymphocyte comprising an artificial celldeath polypeptide, wherein said artificial cell death polypeptide is atransmembrane protein comprising an extracellular domain that comprisesa B-cell activating factor (BAFF) epitope or mimotope, a transmembranedomain, and an intracellular domain comprising an apoptosis-inducingdomain, wherein said apoptosis-inducing domain is caspase 3, caspase 8or caspase 9, wherein said artificial cell death polypeptide isdimerizable using an anti-BAFF antibody that binds to said BAFF epitopeor mimotope, and wherein when said antibody dimerizes said artificialcell death polypeptide, an apoptosis-inducing signal is generated insaid T lymphocyte.
 2. The T lymphocyte of claim 1, wherein said antibodyhas been approved by the United States Food and Drug Administration forany use.
 3. The T lymphocyte of claim 1, wherein said antibody isbelimumab.
 4. The T lymphocyte of claim 3, wherein said extracellulardomain comprises a BAFF epitope.
 5. The T lymphocyte of claim 3, whichfurther comprises a chimeric antigen receptor (CAR) that recognizes anantigen on a tumor cell.
 6. The T lymphocyte of any of claim 1, whichfurther comprises a chimeric antigen receptor (CAR) that recognizes anantigen on a tumor cell.
 7. The T lymphocyte of claim 6, wherein saidtumor cell is a cell in a solid tumor.
 8. The T lymphocyte of claim 6,wherein said tumor cell is a cell of a blood cancer.
 9. The T lymphocyteof claim 6, wherein said antigen is Her2, prostate stem cell antigen(PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancerantigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membraneprotein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin,cytokeratin, desmin, glial fibrillary acidic protein (GFAP), grosscystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A(melanoma antigen recognized by T lymphocytes; MART-1), myo-D1,muscle-specific actin (MSA), neurofilament, neuron-specific enolase(NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin,thyroid transcription factor-1, or the dimeric form of the pyruvatekinase isoenzyme type M2 (tumor M2-PK).
 10. A method of killing a Tlymphocyte, wherein said T lymphocyte comprises a plurality ofartificial cell death polypeptides, wherein each of said artificial celldeath polypeptides is a transmembrane protein comprising anextracellular domain that comprises a B-cell activating factor (BAFF)epitope or mimotope, a transmembrane domain, and an intracellular domaincomprising an apoptosis-inducing domain, wherein said apoptosis-inducingdomain is caspase 3, caspase 8 or caspase 9, wherein each of saidartificial cell death polypeptides is dimerizable using an anti-BAFFantibody that binds to said BAFF epitope or mimotope, and wherein whensaid antibody dimerizes said artificial cell death polypeptides, anapoptosis-inducing signal is generated in said T lymphocyte, comprisingcontacting said T lymphocyte with an amount of said antibody sufficientfor said plurality of artificial cell death polypeptides to dimerize andgenerate an aggregate apoptosis-inducing signal sufficient to kill saidT lymphocyte.
 11. The method of claim 10, wherein said antibody has beenapproved by the United States Food and Drug Administration for any use.12. The method of claim 10, wherein said antibody is belimumab.
 13. Themethod of claim 12, wherein said extracellular domain comprises a BAFFepitope.
 14. The method of claim 12, wherein said T lymphocyte furthercomprises a chimeric antigen receptor (CAR) that recognizes an antigenon a tumor cell.
 15. The method of claim 10, wherein said T lymphocytefurther comprises a chimeric antigen receptor (CAR) that recognizes anantigen on a tumor cell.
 16. The method of claim 15, wherein saidantigen is Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein(AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125),CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelialtumor antigen (ETA), tyrosinase, melanoma- associated antigen (MAGE),CD34, CD45, CD99, CD 1 17, chromogranin, cytokeratin, desmin, glialfibrillary acidic protein (GFAP), gross cystic disease fluid protein(GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognizedby T lymphocytes; MART-1), myo-Dl, muscle-specific actin (MSA),neurofilament, neuron-specific enolase (NSE), placental alkalinephosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, or the dimeric form of the pyruvate kinase isoenzyme type M2 (tumorM2-PK).
 17. The method of claim 10, wherein the method is for killingthe T lymphocyte in an individual to whom the T lymphocyte has beenadministered, wherein the contacting of said T lymphocyte with an amountof said antibody comprises administering the amount of said antibody tothe individual, and wherein the method is for use in the event that theadministration of the T lymphocyte causes any unwanted or deleteriouseffects in the patient receiving the T lymphocyte or in the event thatthe presence of the T lymphocyte in the patient is no longer necessary.18. The method of claim 12, wherein the method is for killing the Tlymphocyte in an individual to whom the T lymphocyte has beenadministered, wherein the contacting of said T lymphocyte with an amountof said antibody comprises administering the amount of said antibody tothe individual, and wherein the method is for use in the event that theadministration of the T lymphocyte causes any unwanted or deleteriouseffects in the patient receiving the T lymphocyte or in the event thatthe presence of the T lymphocyte in the patient is no longer necessary.